CN106636157A - Mutation alpha-whey protein expression vector, preparation method and application thereof - Google Patents

Abstract

The invention discloses a mutant α-lactalbumin expression vector, the nucleotide sequence of the mutant α-lactalbumin expression vector is shown in SEQ ID NO.1, the expression vector contains human α-lactalbumin The TGT sequence corresponding to the 73rd cysteine of the protein gene fragment is mutated into the GCT sequence corresponding to the alanine, and at the same time, ATT, CAG, TCA, AGG and AAC are sequentially inserted between the 71st amino acid and the 72nd amino acid . The invention also discloses a method for preparing HAMLET by using the expression vector of mutant α-lactalbumin. This method solves the problem that in the existing method of preparing HAMLET through ion exchange chromatography column and metal nickel chelation column, due to the cleaning effect of EDTA on NTA metal nickel chelation column, the production steps are increased, and the risk of protein denaturation and degradation is increased. It is easy to cause the failure of HAMLET preparation. A rapid and large-scale preparation method of HAMLET was established.

Description

A mutant α-lactalbumin expression vector, preparation method and application thereof

technical field

The invention belongs to the technical field of biological preparations, and in particular relates to a mutant α-lactalbumin expression vector, a preparation method and an application thereof.

Background technique

HAMLET (Humanα-Lactalbumin Made Lethal to Tumor Cells) is a compound with human α-lactalbumin as the main component, which can cause a large number of tumors to die. HAMLET was discovered accidentally in the study of antibacterial effect of human milk, and then proved to be an effector molecule with strong killing effect on tumor cells. In studies using different cell lines, it was found that HAMLET had the effect of selectively inducing apoptosis of transformed cells, some embryonic cells and lymphocytes, but had no significant toxic effect on mature differentiated cells. Further studies have shown that HAMLET has a broad-spectrum anti-tumor effect and can kill more than 40 tumor cell lines from different tissue sources. In animal models and pre-clinical trials, HAMLET has good curative effect on human glioma, skin papilloma and bladder cancer, and has no obvious side effects. In view of the potential clinical application prospects of HAMLET in tumor therapy, current HAMLET research has begun to focus on its structural characteristics, the mechanism of inducing tumor cell death, and the optimization of its large-scale production process.

At present, the structure of HAMLET has been clarified, and it is mainly composed of human α-lactalbumin (Human α-Lactalbumin, HLA) and oleic acid (oleic acid C18:1, OA) in a certain proportion. HLA structure research has been very clear, it is composed of 123 amino acids molecular weight 14.2kDa acidic calcium binding protein, mainly has two structural domains: a large α-helical domain contains 4 α-helices (5-11, 23 -34, 86-98 and 106-110 amino acids) and a domain consisting of three β-sheets (41-43, 48-50 and 55-56 amino acids). The two domains are linked by a calcium ion-binding loop (amino acids K79, D82, D84, D87, and D88) and formed by four disulfide bonds (amino acids 6-12, 61-77, 73-91, and 28-111) Structurally stable globular protein. However, this natural HLA can combine with oleic acid to form HAMLET only after losing Ca ²⁺ to form apo-HLA in molten globular structure. The presence of two disulfide bonds (61-77, 73-91) is the key component for the formation of HAMLET for the formation of calcium ion-binding loop and molten globular α-lactalbumin.

The existing method for preparing HAMLET through ion-exchange chromatography column and metal nickel chelation column, prokaryotic expression of rHLA protein containing his tag, after purification by NTA metal nickel chelation column, and then removal of calcium ions by EDTA to obtain apo-rHLA, The dialyzed apo-rHLA was combined with water-soluble oleic acid on the NTA metal nickel chelation column to finally form HAMLET. In this method, due to the cleaning effect of EDTA on the NTA metal nickel chelation column, the production steps of mutant α-lactalbumin are increased, and the risk of protein denaturation and degradation is increased, which easily leads to the failure of HAMLET preparation.

Contents of the invention

The object of the present invention is to provide a kind of mutant α-lactalbumin expression carrier, preparation method and application thereof, solve the existing method for preparing HAMLET by ion-exchange chromatography column and metal nickel chelation column, due to EDTA to NTA The cleaning effect of the metal nickel chelate column increases the production steps of mutant α-lactalbumin, and increases the risk of protein denaturation and degradation, which easily leads to the failure of HAMLET preparation.

The present invention provides a mutant α-lactalbumin expression vector, the nucleotide sequence of the mutant α-lactalbumin expression vector is shown in SEQ ID NO.1.

The present invention also provides a method for preparing a mutant α-lactalbumin expression vector, comprising the following steps:

Step 1, inserting the gene fragment of human α-lactalbumin into the pET30a plasmid to construct the pET30a-HLA plasmid, the gene fragment of human α-lactalbumin is shown in SEQ ID NO.2;

Step 2, Mutation of pET30a-HLA plasmid

Step 2.1, designing primers Mu-F1 and Mu-R1 for preparing the pET30a-HLA expression vector, and phosphorylation-modifying the 5' ends of the primers Mu-F1 and Mu-R1 respectively;

The sequence of Mu-F1 is:

5'-TCAGTCAAGGAACATCGCTGACATCTCCTGTGAC-3';

The sequence of Mu-R1 is:

5'-ATGTTCCTTGACTGAGGGACCTGGCTGCTCTTGC-3';

Step 2.2, using the primers Mu-F1 and Mu-R1 in step 2.1, and using a point mutation kit to convert the TGT sequence corresponding to the 73rd cysteine of the human α-lactalbumin gene fragment in the pET30a-HLA plasmid Mutate to the GCT sequence corresponding to alanine, and at the same time insert ATT, CAG, TCA, AGG and AAC between the 71st amino acid and the 72nd amino acid of the gene fragment of human α-lactalbumin to obtain the mutated PCR product;

In step 2.3, the mutated PCR product is recovered, and the mutated PCR product is subjected to a self-ligation reaction to recover the mutant α-lactalbumin expression vector.

The present invention also provides the application of the mutant α-lactalbumin expression vector in preparing HAMLET.

The present invention also provides a method for preparing HAMLET using the above mutant α-lactalbumin expression vector, comprising the following steps:

Step (1), transfer the mutant α-lactalbumin expression vector into Escherichia coli competent cells BL21, express the mutant α-lactalbumin, and collect the BL21 cell precipitate containing the mutant α-lactalbumin ;

In step (2), add 1×PBS buffer solution to resuspend the BL21 cell pellet, transfer it to a container, put it on ice for 30 minutes, ultrasonically break the bacteria until the liquid is clear, centrifuge, and collect the cell debris pellet;

In step (3), resuspend the cell debris pellet with GuMCAC-0 buffer, place at room temperature for 2-4h, then centrifuge at 16000-18000g for 30min at 4°C, collect the supernatant to obtain a solution containing mutant α-lactalbumin, Then refold the mutant α-lactalbumin to obtain a refolded protein solution;

Each liter of GuMCAC-0 buffer is made of the following components: 20 mmol of Tris-HCl, 0.5 mol of NaCl, 6 mol of guanidine hydrochloride, 100 ml of glycerol, and the balance is deionized water, and the pH is adjusted to 7.9 with concentrated HCl.

Step (4), the preparation of HAMLET

Step (4.1), the refolding protein solution is passed through the metal nickel chelating column with a flow rate of 10-15ml/h;

Step (4.2), wash metal nickel chelation column with MCAC-0, flow rate is 20-30ml/h;

Step (4.3), wash metal nickel chelation column with the oleic acid solution of 10-15ml, flow velocity is 10-20ml/h, then leave standstill 2-4h, rinse metal nickel chelation column with MCAC-0 of 10ml at last;

Step (4.4), segmental elution: take 10ml of the following buffers respectively, and wash the metal nickel chelate column with the following buffers in turn: MCAC-20, MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-200, MCAC-500, and the flow rate is 10-20ml/h, and collect the rinsing solution of MCAC-100 and MCAC-200;

Among them, each liter of MCAC-0 is made of the following components: 8mmol of Na ₂ HPO ₄ , 137mmol of NaCl, 2mmol of KH ₂ PO ₄ , 2.7mmol of KCl, 20mmol of Tris-HCl, 50ml of glycerol, and the balance is Deionized water, adjust pH to 6.8 with concentrated HCl;

Each liter of MCAC-500 is made of the following components: 8mmol of Na ₂ HPO ₄ , 137mmol of NaCl, 2mmol of KH ₂ PO ₄ , 2.7mmol of KCl, 20mmol of Tris-HCl, 50ml of glycerol, 0.5mol of imidazole, The balance is deionized water, and the pH is adjusted to 6.8 with concentrated HCl;

MCAC-200 is made by mixing MCAC-0 and MCAC-500 in a volume ratio of 3:2; MCAC-100 is made by mixing MCAC-0 and MCAC-200 in a volume ratio of 1:1; MCAC-80 is made by mixing MCAC- 0 and MCAC-100 are mixed in a volume ratio of 1:4; MCAC-60 is made by mixing MCAC-0 and MCAC-80 in a volume ratio of 3:4; MCAC-40 is made by mixing MCAC-0 and MCAC-80 by 1:1 volume ratio mixed; MCAC-20 is made by mixing MCAC-0 and MCAC-40 according to 1:1 volume ratio;

Step (4.5), add the rinsing solution of MCAC-100 or MCAC-200 into the first dialysis bag, then place the first dialysis bag in the dissolution buffer, and dialyze at 4°C for 24-28h to obtain HAMLET dialysate , wherein the dissolving buffer is a Tris-HCl solution of 20mmol/L pH=8.0;

In step (4.6), the HAMLET dialysate is vacuum freeze-dried to obtain HAMLET.

Preferably, the renaturation of the mutant α-lactalbumin specifically includes the following steps:

Add the solution containing mutant α-lactalbumin into the second dialysis bag, then place the dialysis bag in 4M refolding buffer, and dialyze at room temperature for 24-28h, during which the 4M refolding buffer is magnetically stirred at 1000rpm, and then pour out 1 /2 volume of 4M refolding buffer, dropwise add refolding buffer without guanidine hydrochloride, continue dialysis at room temperature for 24-28h;

Wherein, the volume ratio of the guanidine hydrochloride-free refolding buffer to the remaining 4M refolding buffer is 1:1;

Each liter of 4M refolding buffer is made of the following components: 10mmol of Tris-HCl, 1mmol of CaCl ₂ , 100mmol of KCl, 10mmol of reduced glutathione, 1mmol of oxidized glutathione, 200ml of glycerol , 4mol guanidine hydrochloride, the balance is deionized water, and the pH is adjusted to 8.0 with concentrated HCl.

Each liter of guanidine hydrochloride-free refolding buffer is made of the following components: 10 mmol of Tris-HCl, 1 mmol of CaCl ₂ , 100 mmol of KCl, 10 mmol of reduced glutathione, 1 mmol of oxidized glutathione, 200ml of glycerin, the balance is deionized water, and the pH is adjusted to 8.0 with concentrated HCl.

Move the second dialysis bag to 500ml dissolution buffer, and dialyze at 4°C for 24-28h to obtain a refolding protein solution, wherein the dissolution buffer is 20mmol/L Tris-HCl solution with pH=8.0.

Preferably, in the method for preparing HAMLET from the above-mentioned mutant α-lactalbumin expression vector, the refolding protein solution in step (4.1) passes through the metal nickel chelating column at a flow rate of 10ml/h;

Preferably, in the method for preparing HAMLET from the above-mentioned mutant α-lactalbumin expression vector, the step (4.2) washes the metal nickel chelate column with MCAC-0, and the flow rate is 20ml/h.

Preferably, in the method for preparing HAMLET from the above-mentioned mutant α-lactalbumin expression vector, in step (4.3), wash the metal nickel chelate column with 10ml of oleic acid solution, the flow rate is 10ml/h, and then stand for 2h.

Preferably, in the method for preparing HAMLET from the above mutant α-lactalbumin expression vector, the flow rate in step (4.4) is 15ml/h.

Compared with the prior art, the present invention has the following advantages:

1. The mutant HLA (mu-HLA) obtained by expressing the mutant α-lactalbumin expression vector can form a structure similar to Apo-HLA, which can be directly combined with oleic acid to form HAMLET, thereby establishing a rapid and large-scale preparation method for HAMLET;

2. The HAMLET prepared by the present invention has stronger tumor cell killing activity than the HAMLET (native-HAM) prepared by native-HLA;

3. The HAMLET prepared in the present invention can bind more OA so as to enhance its activity.

Description of drawings

Fig. 1 is the nucleotide sequence alignment result of mu-HLA and native-HLA;

Fig. 2 is the amino acid sequence alignment result of mu-HLA and native-HLA;

Figure 3 is a comparative analysis of the spatial structure of native-HLA and mu-HLA;

Figure 4 is the activity analysis of mu-HAM and native-HAM;

Figure 5 is the analysis of the tumor cell killing activity of mu-HAM and native-HAM;

Figure 6 shows the concentration of free fatty acids in different concentrations of mu-HAM and Native-HAM.

detailed description

The present invention will be described in detail below with reference to the drawings and specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

The present invention provides a mutant alpha-lactalbumin expression vector, the base sequence of the mutant alpha-lactalbumin expression vector is shown in SEQ NO.1.

The present invention also provides a method for preparing a mutant α-lactalbumin expression vector, comprising the following steps:

Step 1, inserting the gene fragment of human α-lactalbumin (HLA) into the pET30a plasmid to construct the pET30a-HLA plasmid, the gene fragment of human α-lactalbumin is shown in SEQ ID NO.2,

The pET30a-HLA plasmid is specifically constructed according to the following steps:

Step 1.1, using the Trizol method to extract the total RNA of MCF-7 breast cancer cells (China Peking Union Medical College Cell Center), and the total RNA was reverse transcribed to obtain the cDNA library of MCF-7 cells.

Step 1.2, using the cDNA library of MCF-7 cells as a template, PCR amplification to obtain the nucleotide sequence for encoding the mature peptide chain of human α-lactalbumin, and then double digestion with Nde I and Xhol I to obtain human The gene fragment of source α-lactalbumin, the gene fragment of described human source α-lactalbumin is shown in SEQ ID NO.2, and the primers for PCR amplification include:

F: 5'-GGGAATTCcatatgAAGCAATTCACAAAATGTGAGCTG-3' (as shown in SEQ ID NO.3);

R: 5'-CCGctcgagCAACTTCTCACAAAAGCCACTGTTCC-3' (as shown in SEQ ID NO.4);

Wherein, the sequences represented by lowercase letters in F and R are enzyme cutting sites.

Step 1.3: Digest the pET30a plasmid (purchased from EMD Biosciences (Novagen)) with Nde I and Xhol I, and connect the gene fragment of human α-lactalbumin shown in SEQ ID NO.2 into NdeI and XholI. The pET30a plasmid was digested with XholI to obtain the pET30a-HLA plasmid. The pET30a-HLA plasmid contains a His fusion C-terminal tag, and its sequence is completely correct after double enzyme digestion identification and DNA sequencing analysis.

Step 2, amplifying the pET30a-HLA plasmid, specifically includes the following steps:

Step 2.1, take 50 μL of E. coli competent cell DH5α in a sterile state into a 1.5 ml Ep tube, add 1 μl of pET30a-HLA plasmid, and place it on ice for 30 minutes.

In step 2.2, place the Ep tube in a water bath at 42°C for 90s, and heat-stimulate the solution in the Ep tube.

Step 2.3, quickly transfer the Ep tube to ice to cool for 3-4min.

In step 2.4, add 400 μl of LB liquid medium to the Ep tube, incubate on a shaker at 37° C. for 1 hour to obtain a resuscitated bacterial liquid, and the LB liquid medium does not contain kanamycin.

Step 2.5, preparing a LB medium plate containing kanamycin, wherein the concentration of kanamycin in the LB medium plate is 100 mg/ml.

In step 2.6, transfer 100 μl of the recovered bacteria solution in step 2.4 to the LB medium plate in step 2.5, spread the recovered bacteria solution flat, and incubate at 37°C for 12-16 hours until a single colony grows. It should be noted that in order to prevent The resuscitated bacteria solution flows back to ensure the best growth state of the microorganisms. After the liquid is absorbed, the plate should be inverted, and then cultured at 37°C.

Step 2.7, aseptically pick up 2 single colonies in 5ml of LB liquid medium containing kanamycin, culture at 37°C for 14 hours to obtain a bacterial liquid containing pET30a-HLA plasmid, wherein kanamycin is contained in LB liquid medium The concentration is 100mg/ml.

In step 2.8, the bacterial solution containing the pET30a-HLA plasmid is extracted with a plasmid extraction kit (OMEGA, plasma MiniKit I, D6943-01). The specific method is shown in the kit manual to obtain the amplified pET30a-HLA expression vector.

It should be noted that step 2 is mainly to enrich the pET30a-HLA plasmid, so as to facilitate the mutation of the pET30a-HLA plasmid, which is a further optimization of the preparation method, and the mutant α-lactalbumin expression vector can still be prepared without this step.

Step 3, Mutation of pET30a-HLA plasmid

Step 3.1, design primers Mu-F1 (as shown in SEQ ID NO.5) and Mu-R1 (as shown in SEQ ID NO.6) that are used to prepare the pET30a-HLA expression vector, and primers Mu-F1 and The 5' end of Mu-R1 was modified by phosphorylation, as shown in Table 1, the above primers Mu-F1 and Mu-R1 were synthesized by Treasure Bio (Dalian) Co., Ltd.

Table 1 PCR primers

Step 3.2, using the primers Mu-F1 and Mu-R1 in step 3.1, using a point mutation kit (ThermoScientific Phusion Site-Directed Mutagenesis kit, F-541) to generate the human α-lactalbumin gene fragment in the pET30a-HLA plasmid The TGT sequence corresponding to the 73rd cysteine was mutated into the GCT sequence corresponding to the alanine, and at the same time, ATT was sequentially inserted between the 71st amino acid and the 72nd amino acid of the human α-lactalbumin gene fragment ( Isoleucine), CAG (glutamine), TCA (serine), AGG (arginine) and AAC (asparagine), to obtain the mutated PCR product, the specific point mutation experiment according to the point mutation kit (Thermo Scientific Phusion Site-DirectedMutagenesis kit, F-541) instructions, the PCR reaction system for specific point mutations is shown in Table 2 and the PCR reaction conditions for point mutations are shown in Table 3.

The role of primers Mu-F1 and Mu-R1 is to mutate the TGT sequence corresponding to the 73rd cysteine of the human α-lactalbumin gene fragment in the pET30a-HLA plasmid to the GCT sequence corresponding to alanine , while sequentially inserting isoleucine, glutamine, serine, arginine and asparagine between the 71st amino acid and the 72nd amino acid of the human α-lactalbumin gene fragment.

Table 2 PCR reaction system

Table 3 PCR reaction program

pre-denatured loop condition number of cycles extend save 98℃, 30sec 98°C, 10sec; 72°C, 30sec; 72°C, 30sec 25 72℃, 10min 4°C

Step 3.3, detection and recovery of mutated PCR products

Take 20 μl of the mutated PCR product in step 3.2, add 3 μl of TaKaRa DNA sample buffer, mix well and spot on 1% agarose gel (containing EB) prepared with 0.5×TBE buffer, and spot 3 μl of standard molecular weight at the same time For molecular weight reference. Electrophoresis was performed at a voltage of 5V/cm. After 60min, the gel was taken out and the amplification effect was observed under ultraviolet light.

The gel block containing the target PCR product was cut from the agarose gel, and the mutated PCR product was recovered with AxyPrep DNA Gel Extraction kit (03213KE1).

Step 3.4, self-ligation reaction of the mutated PCR product

The mutated PCR product was subjected to a self-ligation reaction at 25° C. for 5 minutes to obtain a ligated product, which was the mutant α-lactalbumin expression vector. Wherein the connection reaction system is shown in Table 4.

Table 4 Ligation reaction system

A method for preparing HAMLET from the above mutant α-lactalbumin expression vector is provided below, which specifically includes the following steps:

Step (1), transfer the mutant α-lactalbumin expression vector into Escherichia coli competent cells BL21, express the mutant α-lactalbumin, and collect the BL21 cell precipitate containing the mutant α-lactalbumin .

In step (1.1), the mutant pET30a-HLA expression vector is transformed into Escherichia coli competent cells BL21 to obtain transformed bacteria.

In step (1.2), inoculate the transformed bacteria in 10 ml of LB liquid medium containing kanamycin, culture with shaking at 37° C. for 10 h, wherein the concentration of kanamycin in the LB liquid medium is 10 μg/ml, to obtain a culture.

In step (1.3), 10 ml of the culture was transferred to 1 L of LB liquid medium containing kanamycin, cultured with shaking at 37° C. until OD600 = 0.6, and the culture time was 3-4 hours.

In step (1.4), add IPTG (isopropylthiogalactopyranoside) to the culture medium in step (1.3), and make the final concentration of IPTG in the culture medium 0.2 mol/L, and continue culturing at 37°C for 3h.

In step (1.5), centrifuge at 5000 rpm at 4°C for 5 minutes, and discard the supernatant to obtain a BL21 cell pellet, which contains mutant α-lactalbumin.

Step (2), extracting the mutated α-lactalbumin in the BL21 bacterial cell precipitation

Step (2.1), add 30ml 1×PBS buffer to resuspend the BL21 bacterial pellet, transfer it into a 100ml beaker, put it on ice for 30min, ultrasonically disrupt the bacteria for 2-4h, until the liquid is clear, centrifuge at 16000g for 30min at 4°C, collect Bacterial fragments were precipitated, and the conditions for ultrasonic destruction were: ultrasonic power 350W, each ultrasonic time was 6s, and the interval between two adjacent ultrasonic waves was 9s, and the total time for ultrasonic destruction was 2-4h.

In step (2.2), the cell debris pellet is washed with 20ml of washing buffer for 3 times, and after each wash, centrifuge at 12000g for 30min at 4°C to collect the precipitate to obtain cell debris, wherein each liter of washing buffer consists of the following components: Preparation: 20mmol of Tris, 1mmol of EDTA and 10ml of Triton X-100, the balance is deionized water, pH 8.0. It should be noted that in the process of preparing HAMLET, step (2.2) can also be omitted.

Step (2.3), resuspend the cell fragments with 10ml GuMCAC-0 buffer, and dissolve the cell fragments completely, place at room temperature for 2h, then centrifuge at 16000g for 30min at 4°C, collect the supernatant, and obtain the protein containing mutant α-lactalbumin solution.

Among them, every liter of GuMCAC-0 buffer is made of the following components: 20mmol of Tris-HCl, 0.5mol of NaCl, 6mol of guanidine hydrochloride, 100ml of glycerin, and the balance is deionized water, and the pH is adjusted to 7.9 with concentrated HCl .

Step (2.4), carrying out the renaturation of the mutant α-lactalbumin with the solution containing the mutant α-lactalbumin, comprises the following steps:

Step (2.4.1), adding the solution containing mutant α-lactalbumin into the second dialysis bag, wherein the molecular weight cut-off of the second dialysis bag is 3KD, and then placing the second dialysis bag in 4M refolding buffer, Dialyze at room temperature for 24 hours, during which the 4M refolding buffer is magnetically stirred at 1000rpm, then pour out 1/2 volume of the 4M refolding buffer, add dropwise the refolding buffer without guanidine hydrochloride, and continue the dialysis at room temperature for 24-28 hours, of which, no The volume ratio of guanidine hydrochloride refolding buffer to the remaining 4M refolding buffer is 1:1;

Each liter of 4M refolding buffer is made of the following components: 10mmol of Tris-HCl, 1mmol of CaCl ₂ , 100mmol of KCl, 10mmol of reduced glutathione, 1mmol of oxidized glutathione, 200ml of glycerol , 4mol guanidine hydrochloride, the balance is deionized water, and the pH is adjusted to 8.0 with concentrated HCl.

Each liter of guanidine hydrochloride-free refolding buffer is made of the following components: 10 mmol of Tris-HCl, 1 mmol of CaCl ₂ , 100 mmol of KCl, 10 mmol of reduced glutathione, 1 mmol of oxidized glutathione, 200ml of glycerin, the balance is deionized water, and the pH is adjusted to 8.0 with concentrated HCl.

In step (2.4.2), the second dialysis bag was moved to 500ml of dissolving buffer, and dialyzed at 4°C for 24-28h to obtain a refolding protein solution, wherein the dissolving buffer was a 20mmol/L Tris-HCl solution with pH=8.0.

Step (3), the preparation of HAMLET

Step (3.1), the refolding protein solution is passed through the metal nickel chelate column with a flow rate of 10ml/h, and the preparation method of the metal nickel chelate column is as follows: prepare a 1.5 * 20cm (20ml biobad) Ni-NTA column, Add 2.5ml metal nickel chelate agarose gel, let the liquid just flow to the top of the column bed, and wash the column with the following liquids in sequence: deionized water (10ml) equivalent to 3 times the column bed volume; equivalent to 3 times the column bed volume bed volume of MCAC-0 (10ml); it should be noted that the refolded protein solution was passed through the metal nickel chelate column at a flow rate of 10-15ml/h.

In step (3.2), wash the metal nickel chelate column with 10ml MCAC-0, and the flow rate is 20ml/h.

Step (3.3), wash metal nickel chelation column with the oleic acid solution of 10ml, and flow velocity is 10ml/h, stand 2h (preferably standing time is 2h), wash metal nickel chelation with 10ml MCAC-0 damping fluid column.

Wherein, every liter of oleic acid solution is prepared according to the following method: the oleic acid of 30ml is dissolved in the ethanol of 50ml, then is settled to 1000ml with the Tris-HCl solution of 20mmol/L, adjusts to pH= with 5mol/L NaOH solution at last 8.0, vortex to mix.

Step (3.4), segmental elution: take 10ml of the following buffers respectively, and wash the metal nickel chelate column with the following buffers in sequence, including: MCAC-20, MCAC-40, MCAC-60, MCAC-80, MCAC- 100, MCAC-200, MCAC-500, collect the rinsing liquid of MCAC-100 and the rinsing liquid of MCAC-200 respectively, and the flow rate is 15ml/h.

Each liter of MCAC-0 is made of the following components: 8mmol of Na ₂ HPO ₄ , 137mmol of NaCl, 2mmol of KH ₂ PO ₄ , 2.7mmol of KCl, 20mmol of Tris-HCl, 50ml of glycerin, and the balance is deionized water, and adjust the pH to 6.8 with concentrated HCl.

Each liter of MCAC-500 is made of the following components: 8mmol of Na ₂ HPO ₄ , 137mmol of NaCl, 2mmol of KH ₂ PO ₄ , 2.7mmol of KCl, 20mmol of Tris-HCl, 50ml of glycerol, 0.5mol of imidazole, The balance was deionized water, and the pH was adjusted to 6.8 with concentrated HCl.

MCAC-200 is made by mixing MCAC-0 and MCAC-500 in a volume ratio of 3:2; MCAC-100 is made by mixing MCAC-0 and MCAC-200 in a volume ratio of 1:1; MCAC-80 is made by mixing MCAC- 0 and MCAC-100 are mixed in a volume ratio of 1:4; MCAC-60 is made by mixing MCAC-0 and MCAC-80 in a volume ratio of 3:4; MCAC-40 is made by mixing MCAC-0 and MCAC-80 by 1:1 volume ratio is mixed; MCAC-20 is made by mixing MCAC-0 and MCAC-40 according to 1:1 volume ratio.

Step (3.5), add the rinsing solution of MCAC-100 or the rinsing solution of MCAC-200 in the first dialysis bag, wherein the molecular weight cut-off of the first dialysis bag is 3KD, then place the first dialysis bag in 500ml of dissolution buffer , dialyzed at 4° C. for 24 hours to obtain HAMLET dialysate, wherein the dissolution buffer was 20 mmol/L Tris-HCl solution with pH=8.0.

It should be noted that any rinsing solution of MCAC-100 or MCAC-200 can be used to prepare HAMLET.

In step (3.7), the HAMLET dialysate is vacuum freeze-dried at -30°C to obtain HAMLET.

It should be noted that the concentrated HCl mentioned in the present invention is all commercially available concentrated HCl.

HLA and oleic acid can be non-covalently combined, but to form active HAMLET, HLA needs to be formed into a molten globule state, that is, Ca ²⁺ is removed by EDTA or heating to form Apo-HLA, Apo-HLA Combined with oleic acid to form HAMLET. Due to the change of the Ca ²⁺ binding domain, the mutant HLA in the present invention can directly combine with oleic acid to form active HAMLET. The specific performance is:

1. The mutant α-lactalbumin (mu-HLA) expressed by the mutant α-lactalbumin expression vector can form a structure similar to Apo-HLA, and directly combine with oleic acid to form HAMLET.

In order to explore the differences between mu-HLA and human α-lactalbumin gene fragments (native-HLA), the nucleotide and amino acid sequences of mu-HLA and native-HLA were compared, and the results are shown in Figure 1 and Figure 2, respectively. As shown, the 73rd amino acid of the native-HLA was changed, and five amino acid sequences were added between the 71st and 72nd amino acids to form mu-HLA.

Their structures were analyzed by ANS binding experiments: 100 μl of 0.2 mg/ml samples were mixed with 50-fold molar concentration of hydrophobic dye ANS, and the binding reaction was carried out at 37°C for 1 h in the dark. The ANS bound to protein can be excited by fluorescence at 365nm, and its emission spectrum in the range of 400-600nm was recorded by Spectra Max M2 spectrophotometer, and processed by SoftMax 5.2 software to draw the spectral curve, the results are shown in Figure 3.

The results in Figure 3 show that the ANS binding activity of mu-HLA is significantly higher than that of native-HLA, making the maximum emission wavelength and peak value of ANS similar to that of apo-HLA, indicating that mu-HLA has a looser structure than mu-HLA and has similar apo - The structure of HLA, which can expose more hydrophobic regions, directly binds with OA to form HAMLET.

2. The HAMLET (mu-HAM) prepared by the present invention has stronger tumor cell killing activity than the HAMLET (native-HAM) prepared by native-HLA

U87MG glioma cells and mouse primary glial cells (PAM) were digested with trypsin, seeded in 96-well plates at 10 ⁵ cells/well, and cultured overnight at 37°C in 5% CO ₂ until the cells were attached. wall, replace serum-free DMEM medium, incubate for 30min, add 1mg/ml mu-HLA, 1mg/ml native-HLA, 0.2μg/ml OA, 0.2mg/ml native-HAM, 0.2mg/ml mu-HAM Or add the control reagent to each group of samples and mix well. After 1 h, FBS was added to a final concentration of 10%. Continue culturing for 24 hours, add MTS reagent, incubate at 37°C for 4 hours, dissolve and dilute the sample, and detect the photometric absorbance at a wavelength of 490nm.

U87MG glioma cells were treated with 200 μg/ml mu-HAM and 200 μg/ml native-HAM, and the killing effect of mu-HAM on U87MG cells was detected by MTS assay, the results are shown in Figure 4. The results showed that the survival rate of U87MG cells treated with mu-HAM (25%) was significantly lower than that of mu-HLA, native-HLA and OA treatment groups (80-90%), and also significantly lower than that of native-HAM The cell survival rate (45%) showed that the activity of mu-HAM prepared with mu-HLA was stronger than that of native-HAM.

At the same time, it can be seen from Figure 5 that the killing effect of different concentrations of mu-HAM and native-HAM on U87MG glioma cells was dose-dependent (two lines with a downward trend in Figure 5), while the effect on small The primary mouse glial cells had no obvious cytotoxicity (the two lines with a trend close to the level in Figure 5), indicating that mu-HAM and native-HAM prepared with mu-HLA had selective tumor killing activity, while mu-HAM The cytotoxicity of 50, 100, 200, 300μg/ml concentration was significantly higher than that of native-HAM.

3. The HAMLET prepared by the present invention can bind more OA to enhance its activity

The biological activity of HAMLET is closely related to the binding amount of OA. In order to detect the protein-bound OA content, the protein was degraded by heating at 100°C for 10 minutes, and after cooling to room temperature, the protein was precipitated with 1ml-20°C cold ethanol. After centrifugation, a 333-fold diluted 20 mM Tris-HCl solution was added to the supernatant. According to the method of the human FFA enzyme-linked immunosorbent assay kit, different concentrations of mu-HAM and Native-HAM were detected and analyzed, and the results are shown in Figure 6. The results show that the free fatty acid concentration of mu-HAM in different concentrations is higher than that of Native-HAM, indicating that it combines more OA, that is, the mu-HLA of the present invention has a higher binding rate with OA, which can enhance the biological activity of HAMLET.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (9)

1. it is a kind of to be mutated alpha lactalbumin expression vector, it is characterised in that the nucleosides of the mutation alpha lactalbumin expression vector Acid sequence is as shown in SEQ ID NO.1.

2. it is a kind of mutation alpha lactalbumin expression vector preparation method, it is characterised in that comprise the following steps：

Step 1, inserts the genetic fragment of people source alpha lactalbumin in pET30a plasmids, constructs pET30a-HLA plasmids, institute The genetic fragment of people source alpha lactalbumin is stated as shown in SEQ ID NO.2；

Step 2, the mutation of pET30a-HLA plasmids

Step 2.1, is designed for preparing the primer Mu-F1 and Mu-R1 of pET30a-HLA expression vectors, and respectively to primer Mu- 5 ' the ends of F1 and Mu-R1 carry out phosphorylation modification；

The sequence of Mu-F1 is：

5’-TCAGTCAAGGAACATCGCTGACATCTCCTGTGAC-3’；

The sequence of Mu-R1 is：

5’-ATGTTCCTTGACTGAGGGACCTGGCTGCTCTTGC-3’；

Step 2.2, using the primer Mu-F1 and Mu-R1 of step 2.1, and using point mutation kit by pET30a-HLA plasmids The corresponding TGT series jumps of 73rd cysteine of the genetic fragment of middle people source alpha lactalbumin are the corresponding GCT of alanine Sequence, while being sequentially inserted between the 71st amino acids and the 72nd amino acids of the genetic fragment of people source alpha lactalbumin ATT, CAG, TCA, AGG and AAC, the PCR primer after being mutated；

Step 2.3, reclaims the PCR primer after mutation, and the PCR primer after mutation is carried out into itself coupled reaction, and recovery is obtained Mutation alpha lactalbumin expression vector.

3. application of the mutation alpha lactalbumin expression vector according to claim 1 in HAMLET is prepared.

4. the method that the mutation alpha lactalbumin expression vector described in a kind of utilization claim 1 prepares HAMLET, its feature exists In comprising the following steps：

Step (1), the mutation alpha lactalbumin expression vector is proceeded in competent escherichia coli cell BL21, is mutated The expression of alpha lactalbumin, and collect the BL21 bacterial sediments containing mutation alpha lactalbumin；

Step (2), plus 1 × the resuspended BL21 bacterial sediments of PBS, in moving into container, is placed on 30min on ice, carrying out ultrasonic bacteria breaking Until liquid is limpid, and centrifugation, collects thalline fragment precipitation；

Step (3), with the resuspended bacterial chip precipitation of GuMCAC-0 buffer solutions, room temperature places 2-4h, then in 4 DEG C, 16000- 18000g is centrifuged 30min, collects supernatant, obtains the solution containing mutation alpha lactalbumin, then carries out mutation alpha lactalbumin Renaturation, obtains recombinant protein solution；

Every liter of GuMCAC-0 buffer solution is made up of following components：The hydrochloric acid of the NaCl of the Tris-HCl of 20mmol, 0.5mol, 6mol Guanidine, the glycerine of 100ml, balance of deionized water adjusts pH to 7.9 with dense HCl；

Step (4), the preparation of HAMLET

Step (4.1), metallic nickel chelate column is crossed by recombinant protein solution with the flow velocity of 10-15ml/h；

Step (4.2), with MCAC-0 metallic nickel chelate column is rinsed, and flow velocity is 20-30ml/h；

Step (4.3), with the oleic acid solutions of 10-15ml metallic nickel chelate column is rinsed, and flow velocity is 10-20ml/h, then stands 2- 4h, finally rinses metallic nickel chelate column with the MCAC-0 of 10ml；

Step (4.4), stepwise elution：10ml following buffers are taken respectively, and successively metallic nickel is washed using following buffer chelate Post：MCAC-20, MCAC-40, MCAC-60, MCAC-80, MCAC-100, MCAC-200, MCAC-500, and flow velocity is 10- 20ml/h, and collect the flushing liquor of MCAC-100 and the flushing liquor of MCAC-200；

Wherein, every liter of MCAC-0 is made up of following components：The Na of 8mmol₂HPO₄, the NaCl of 137mmol, the KH of 2mmol₂PO₄, The glycerine of the Tris-HCl of the KCl of 2.7mmol, 20mmol, 50ml, balance of deionized water adjusts pH to 6.8 with dense HCl；

Every liter of MCAC-500 is made up of following components：The Na of 8mmol₂HPO₄, the NaCl of 137mmol, the KH of 2mmol₂PO₄, The glycerine of the Tris-HCl of the KCl of 2.7mmol, 20mmol, 50ml, the imidazoles of 0.5mol, balance of deionized water uses dense HCl Adjust pH to 6.8；

MCAC-200 is mixed by MCAC-0 and MCAC-500 by 3: 2 volume ratio；MCAC-100 is by MCAC-0 and MCAC- 200 are mixed by 1: 1 volume ratio；MCAC-80 is mixed by MCAC-0 and MCAC-100 by 1: 4 volume ratio； MCAC-60 is mixed by MCAC-0 and MCAC-80 by 3: 4 volume ratio；MCAC-40 presses 1: 1 by MCAC-0 and MCAC-80 Volume ratio mix；MCAC-20 is mixed by MCAC-0 and MCAC-40 by 1: 1 volume ratio；

Step (4.5), the flushing liquor of the flushing liquor of MCAC-100 or MCAC-200 is added in the first bag filter, then by the One bag filter is placed in dissolving buffer solution, 4 DEG C of dialysis 24-28h, obtains HAMLET dislysates, wherein dissolving buffer solution is The Tris-HCl solution of the pH=8.0 of 20mmol/L；

Step (4.6), by HAMLET dislysate vacuum freeze dryings, obtains HAMLET.

5. the method that mutation alpha lactalbumin expression vector according to claim 4 prepares HAMLET, it is characterised in that institute Mutation alpha lactalbumin renaturation is stated, following steps are specifically included：

Solution containing mutation alpha lactalbumin is added in the second bag filter, then bag filter 4M renaturation buffers is placed in into In, then room temperature dialysis 24-28h, period 1000rpm magnetic agitation 4M renaturation buffer pours out the 4M renaturation buffering of 1/2 volume Liquid, is added dropwise to without guanidine hydrochloride renaturation buffer, continues room temperature dialysis 24-28h；

Wherein, the volume ratio without guanidine hydrochloride renaturation buffer and residue 4M renaturation buffers is 1: 1；

Every liter of 4M renaturation buffer is made up of following components：The CaCl of the Tris-HCl of 10mmol, 1mmol₂, the KCl of 100mmol, The reduced glutathione of 10mmol, the oxidized form of glutathione of 1mmol, the glycerine of 200ml, 4mol guanidine hydrochlorides are balance of to go Ionized water, with dense HCl pH to 8.0 is adjusted；

Per liter is made up without guanidine hydrochloride renaturation buffer of following components：The CaCl of the Tris-HCl of 10mmol, lmmol₂, The reduced glutathione of the KCl of 100mmol, 10mmol, the oxidized form of glutathione of 1mmol, the glycerine of 200ml is balance of Deionized water, with dense HCl pH to 8.0 is adjusted；

Second bag filter is moved into 500ml dissolving buffer solutions, 4 DEG C of dialysis 24-28h obtain recombinant protein solution, wherein dissolving is slow Rush the Tris-HCl solution of the pH=8.0 that liquid is 20mmol/L.

6. the method that mutation alpha lactalbumin expression vector according to claim 4 prepares HAMLET, it is characterised in that step Suddenly recombinant protein solution crosses metallic nickel chelate column with the flow velocity of 10ml/h in (4.1).

7. the method that mutation alpha lactalbumin expression vector according to claim 4 prepares HAMLET, it is characterised in that step Suddenly (4.2) with MCAC-0 rinse metallic nickel chelate column, flow velocity is 20ml/h.

8. the method that mutation alpha lactalbumin expression vector according to claim 4 prepares HAMLET, it is characterised in that step Suddenly metallic nickel chelate column is rinsed with the oleic acid solutions of 10ml in (4.3), flow velocity is 10ml/h, then stands 2h.

9. the method that mutation alpha lactalbumin expression vector according to claim 4 prepares HAMLET, it is characterised in that step Suddenly the flow velocity of (4.4) is 15ml/h.