WO2014075445A1 - First 180bp segment of polyhedrin gene and application thereof - Google Patents

Abstract

Provided are a first 180bp segment of a polyhedrin gene and an application thereof. The nucleotide sequence of the first 180bp segment of the polyhedrin gene is shown as SEQ ID NO:1. The segment is applicable to the expression of a fusion protein. Further provided is an expression carrier, which is formed by multiple cloning sites of a starting carrier being sequentially inserted with the foregoing segment and a protease digestion site segment. The sequence of the first 180bp of the polyhedrin gene in the present invention has more desirable properties than its full-length sequence, and can be connected to other target genes to express a fusion protein, which not only implements the effect of increasing an expression amount, but also increases the solubility of the fusion protein in a condition of the neutral pH value, so as to adapt to an optimal pH value for the function of protease digestion, and facilitate the acquisition of a target protein.

Description

 Polymorphic gene 180 bp fragment and its application

Technical field

 The invention relates to the field of genetic engineering technology, and particularly relates to a 180 bp fragment of a polyhedrin gene and an application thereof. Background technique

 The Bombyx mori Polyhedrin (Poh) gene has a total of 738 nucleotides and encodes a polypeptide of 29 kD consisting of 245 amino acids. It is the major structural component of the polyhedron. The protein crystals composed of polyhedrin proteins have a protective effect on the virions embedded therein, which keeps the virions stable and infective in the natural environment. The polyhedrin gene polh is a super-expressing very late gene. In the late stage of viral infection, when other viral genes and host genes are closed, the gene can continue to express at a high level, and the amount of polyhedrin produced in the infected cells can reach 25 to 50% of the total amount of cellular protein. In the past 20 years, people have been working on the efficient expression mechanism of the polyhedrin gene, and using this property has enabled many foreign genes to be efficiently expressed, especially proteins that are difficult to express, such as membrane proteins. The polyhedrin protein of Bombyx mori nuclear polyhedrosis virus has its unique properties. The polyhedrin expressed in E. coli exists in the form of inclusion bodies under neutral pH conditions, only in alkaline conditions (pH 10.8 and above). It is soluble, and this polyhedrin has the same properties as the natural polyhedrin crystal. Therefore, the target protein gene can be expressed in fusion with one another, and on the other hand, the expression level of the target protein can be increased. On the other hand, a simple fusion protein can be obtained by simple pH gradient elution and differential centrifugation, and then The protease is digested and the pure protein of interest is recovered.

However, when polyhedral fusion foreign protein expression is currently used, since the polyhedron is only soluble under alkaline conditions, the protease reaction is most suitable in the process of digesting the fusion protein with a protease to obtain a separate protein of interest. The pH value is generally neutral (pH 7 to 9), which results in the inability to simply obtain the desired protein by enzymatic cleavage under the optimal conditions of the enzyme, and when the enzyme is cleaved under alkaline conditions, the protease is protease. The activity is lost or the maximum activity is not achieved, and the amount of the target protein obtained is also limited. Summary of the invention In order to solve the above problems, the present invention provides a fragment of a polyhedrin gene of Bombyx mori, which is fused with a gene of interest, and in addition to satisfying a large amount of expression, the solubility property thereof is also changed, and is within a suitable pH range of the protease. It is soluble, which greatly simplifies the digestion and purification steps. The first 180 bp fragment of the polyhedrin gene of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1. The application of the first 180 bp fragment of the above polyhedrin gene in protein fusion expression. An expression vector is constructed by inserting a multiple cloning site of the polyhedrin gene into the first 180 bp fragment of the polyhedrin gene and a protease cleavage site fragment. The fusion protein is expressed and digested with the corresponding protease to obtain the protein of interest. Preferably, the starting vector is a pET series vector, a pcDNA3 series vector, a pFastBacTM series vector, a pKK series vector or a pBAD series vector. Among them, pET-28a of pET series vector is the best. Preferably, the protease cleavage site fragment is a thrombin cleavage site fragment. The nucleotide sequence of the thrombin cleavage site fragment is shown in SEQ ID NO: 2. A fusion protein expression vector is constructed by inserting the first 180 bp fragment of the polyhedrin gene, the protease cleavage site fragment and the target gene fragment in turn at the multiple cloning site of the starting vector. Preferably, the gene of interest is an EGFP (Enhanced Green Fluorescent Protein) gene, a LZM (lysozyme) gene or a MMgT (membrane magnesium transporter protein) gene.

 A genetically engineered bacterium comprising the above fusion protein expression vector. Preferably, the genetically engineered bacteria is Escherichia coli. Compared with the prior art, the present invention has the following beneficial effects:

The present invention finds that the partial sequence has more excellent properties than the full-length sequence based on the polyhedrin gene polh, and can express the fusion protein together with other target genes, thereby not only achieving the effect of increasing the expression amount, but also increasing the fusion protein. The solubility under neutral pH conditions makes it suitable for the optimal pH of the protease digestion function, which greatly facilitates the acquisition of the target protein. DRAWINGS Figure 1 is a map of the pET-28a-polhl80-EGFP plasmid.

 Figure 2 is an electrophoresis map of the PCR product of the first 180pb fragment of the polyhedrin gene; wherein, M: nucleic acid molecular weight standard; 1: target gene PCR product.

 Figure 3 is a PCR amplification diagram of EGFP, M: marker; 1: EGFP amplification product.

 Figure 4 is a diagram showing the identification of a recombinant expression vector, wherein M: nucleic acid molecular weight standard; 1: EGFP gene PCR product; 2: recombinant expression vector pET-28a-polhl80-EGFP was digested with EcoR l^WXho l product strip.

 Figure 5 shows the expression of pET-28a-polhl80-EGFP protein, M: protein molecular weight standard; 1: pET-28a-polhl80-EGFP uninduced; 2: pET-28a-polhl80-EGFP induction; 3: pET-28a-polh -EGFP induction; 4: pET-28a-EGFP induction.

 Figure 6 is a graph showing the solubility analysis of the polhl80-EGFP fusion protein; wherein, 1, 3, 5, 7, 9, 11, 13 are the supernatants at pH 8.0, 8.4, 9.2, 9.6, 10.0, 10.4, 10.8; 2, 4, 6, 8, 10, 12, 14 are precipitates at pH 8.0, 8.4, 9.2, 9.6, 10.0, 10.4, 10.8.

 Figure 7 is an electropherogram of the pH value of the polhl80-EGFP protein solution using Gly; wherein, M: protein molecular weight standard; 5: polhl80-EGFP supernatant (pH 10.8); 6: polhl80-EGFP supernatant (pH 9.0) 7: polhl80-EGFP supernatant (pH 8.0); 8: polhl80-EGFP precipitation (pH 8.0); 9: polh-EGFP supernatant (pH 10.8); 10: polh-EGFP precipitation (pH 10.8) .

 Figure 8 is a graph showing the results of electrophoresis after oxidase digestion of polhl80-EGFP fusion protein, wherein M: protein molecular weight standard; 1: supernatant at pH 7.6; 2: sample after thrombin digestion at pH 7.6 . detailed description

 The present invention will be further described in conjunction with the accompanying drawings and specific embodiments, which are to be understood by those skilled in the art.

 The biomaterials used in the following examples, such as Bombyx mori nuclear polyhedrosis virus, vector pET-28a, plasmids PGEX-4T-1-EGFP and E. coli TGI were purchased from Tefi (Tianjin) Biomedical Technology Co., Ltd. Example 1: Construction of expression vector pET28a-jw/zl80

(1); The gene sequence is known, and any plasmid containing the gene fragment or such as a virus can be used. When other biological materials are used as templates, this embodiment uses the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome as a template to amplify the first 180 bp fragment of the polyhedrin gene. Primer design is as follows: Upstream primer F _: 5'-CGC|GGATCC TGCCGAATTATTCATACAC-3' (SEQ ID NO: 3, Bamli I restriction site); Downstream primer R:

(SEQ ID NO: 4, the coR I restriction site in the box, and the underlined portion is the coagulation site ⁵ )' (2) Amplification of the first 180 bp fragment of the polyhedrin gene

 Using the viral genome as a template, the F and R sequences of the step (1) were subjected to PCR amplification. PCR reaction system: In the centrifuge tube of ΙΟΟμΙ^, add the following components:

 lOxTaq Buffer I 5 μ∑

 2.5 mM dNTPs 5 μL

 Taq DNA polymerase 1.5 μL

 F 1 μL

 R 1 μL

 1 μL

 Sterile double distilled water 35.5 L

 Total volume 50

 After mixing the components, put them into the PCR instrument. The reaction parameters are designed as: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, refolding at 72 °C for 20 s, 30 cycles, 72 °C extended for 3 min. 30 cycles were designed according to the above reaction parameters. After the reaction is completed, the amplified fragment is identified by electrophoresis (see Fig. 2), and the target fragment is recovered by gelatinization. The amplified product was 180 bp in size and sequenced, which was identical to the first 180 bp in GenBank accession number JQ291703.1.

 (3) The gene fragment obtained by the above-obtained PCR product was digested with BamH I and EcoR I into a vector pET-28a double-digested with Βωηΐ!I and EcoR I, and the PCR-amplified gene was ligated to the vector pET. On the -28a, the recombinant vector pET-28a-polhl80 was constructed, and the gene sequence was completely confirmed by restriction analysis.

Example 2: Fusion expression EGFP

(1) Amplification of the EGFP gene sequence For the EGFP gene sequence, see GenBank accession number JQ969017.1, which is a known sequence, any biological material containing the gene fragment can be used as an amplification template. For convenience of operation, this example uses the plasmid pGEX-4T-1 -EGFP as a template. The design of the bow 1 amplification of the EGFP gene fragment.

 The EGFP gene ORF sequence primers designed with Primer5.0 are as follows:

Upstream arch 1 F,: 5'-GCClGAATT^ATGGTGAGCAAGGGCGAGGA -3' (SEQ ID NO: 5, within the box is the EcoR I restriction site);

Downstream arch I R': 5'-CCG|CTCGA TTACTTGTACAGCTCGTCCATG-3' (SEQ ID NO: 6, in the box is the [enzymatic cleavage site). lOxTaq Buffer I 5 μ∑

 2 .5 mM dNTPs 5 μL

 Taq DNA polymerase 1.5 μL

 F, 1.5 μL

 R' 1.5 μL

 1 μL

 Aseptic double distilled water 34.5μ∑

 Total volume 50 μ∑

 The components were added according to the PCR reaction system, mixed, and placed in a PCR machine. The PCR reaction parameters were designed as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, refolding at 72 °C for 50 s, 30 cycles, and extension at 72 °C for 7 min. 30 cycles were designed according to the above reaction parameters. After the reaction is completed, the amplified fragment is identified by electrophoresis (see Fig. 3), and the target fragment is recovered by gelatinization and identified by sequencing.

(2) The gene fragment obtained by digesting the PCR product obtained by EcoR I and 3⁄4 ₀ I was cloned into the above recombinant vector pET-28a-polhl80 digested with EcoR i xho l to make a PCR amplified gene. The recombinant expression vector pET-28a-polhl80-EGFP was constructed by ligation to pET-28a-polhl80 (see Figure 1). The restriction enzyme digestion analysis confirmed that the ligation was completely correct (see Figure 4). The size of the recombinant expression vector was 6287 bp.

 (3) Expression of pET-28a-polhl80-EGFP

The recombinant expression vector pET-28a-polhl80-EGFP was transferred to E. coli TGI, and cultured in an LB plate medium containing 50 μ _§ /ηΛ kanamycin sulfate (Kan), and single colonies were picked, in LB. The culture was continued to expand, and the recombinant plasmid pET-28a-polhl80-EGFP was extracted for PCR and double enzyme digestion, and identified by sequencing. Transfer to E.coli BL21 and add IPTG to a final concentration of 1 mM. The expression was induced at 37 °C. After 4 hours, the cells were collected, centrifuged at 12000 rpm for 10 min, and the precipitate was collected. Resuspended in 5 (^L of lxPBS, then added 5 (^L of 2x loading buffer for sample preparation, and 15 L of sample was taken. 12% of SDS-PAGE protein was electrophoresed, and the empty expression vector pET-28a-EGFP and vector pET-28a-polh-EGFP were used as controls. The results are shown in Fig. 5. After induction, the polhl80-EGFP fusion protein was expressed in a large amount. .

Example 3: Fusion Protein Solubility Verification

 (1) The cells after induction of expression were collected, and the cells were sonicated and centrifuged at 12000 rpm for 10 min. The precipitate was collected, resuspended in a buffer of pH 8.0, stirred at 4 ° C for 30 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken as The supernatant of pH 8.0; the collected precipitate was resuspended in an equal volume of buffer of pH 8.4, stirred at 4 ° C for 30 min, and the resuspension was taken as the precipitation component of the previous pH 8.0, 12,000 rpm. After centrifugation for 10 min, the supernatant was taken as the supernatant of pH 8.4. Similarly, an equal volume of pH 8.8 buffer was added to the upper pellet, stirred at 4 ° C for 30 min, and the resuspended suspension was used as a pH 8.4 precipitation group. The supernatant was centrifuged at 12,000 rpm for 10 min, and the supernatant was taken as a supernatant of pH 8.8; and so on, the supernatant and precipitate components of the fusion protein polhl80-EGFP at different pH conditions were obtained. Samples of different components were taken and analyzed by SDS-PAGE to detect the solubility change of the fusion protein polhl80-EGFP with pH. The formulation of the buffer is shown in Table 1.

 Table 1 Formulation of different pH buffers

pH 0.2 M Gly(mL) 0.2 M NaOH (mL) ddH ₂ 0 (mL) Total volume (mL)

 8.0 15 0.45 44.55 60

 8.4 15 1.06 43.94 60

 8.8 15 1.8 43.2 60

 9.2 15 3.6 41.4 60

 9.6 15 6.72 38.28 60

 10.0 15 9.6 35.4 60

 10.4 15 11.58 33.42 60

 10.8 15 13.65 31.35 60

 The experimental results are shown in Fig. 6. As can be seen from the figure, the fusion protein is mostly in the supernatant in the buffer with a pH of 10.8, and some of it is still in the precipitate.

(2) The supernatant of the above polhl80-EGFP protein having a pH of 10.8 was taken, and 0.2 M of Gly (glycine) was added dropwise thereto while stirring, and the pH of the solution was measured at any time. Meanwhile, the supernatant of the polh-EGFP protein having a pH of 10.8 was used as a control. It was found that the polhl80-EGFP protein solution was adjusted back to 9.0 and there was no turbidity. At 12000 rpm, centrifugation for 10 min, there was no precipitation at the bottom of the centrifuge tube; the pH was further lowered to 8.0, and no turbidity occurred. At 12000 rpm, centrifugation for 10 min, we can see There is a little sediment at the bottom of the tube.

 In the control group, the polh-EGFP protein solution was found to have turbidity at a pH of 10.8. At 12,000 rpm, it was centrifuged for 10 min, and a large amount of precipitate was observed at the bottom of the centrifuge tube.

 The supernatants and precipitates of the polhl80-EGFP fusion protein solution were separately taken for electrophoresis. As shown in Figure 7, when the pH is adjusted to 8.0, the supernatant protein in the supernatant is more than the target protein in the precipitate, which is sufficient to lower the pH of the solution. This pH can be regarded as a neutral buffer pH. value. Example 4: Protease digestion of polhl80-EGFP

 The polhl80-EGFP fusion protein solution of Example 3 was adjusted back to pH 8.0, and the fusion protein was subjected to a digestion reaction in a 1.5 mL centrifuge tube according to the Novagen THROMBIN product specification. The response system is as follows:

 Fusion protein 120μΕ

 1 Ox thrombin digestion buffer (Thrombin cleavage Buffer) 20μL

 Thrombin, Restriction Grade 8μΙ^

ddH ₂ 0 52μ

 The total volume of 20 (^L mixed, 21 °C reaction 2h, sampling and electrophoresis. The results show (see Figure 8), the protein size after digestion is basically the same as the theoretical EGFP size, and the band of the fusion protein is very It can be said that thrombin substantially completely cleaves the EGFP enzyme in the fusion protein. The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. The equivalents and modifications of the present invention are intended to be within the scope of the present invention. The scope of the present invention is defined by the appended claims.

Claims

Claim  A 180 bp fragment of a polyhedrin gene, which is characterized in that the nucleotide sequence is as shown in SEQ ID NO: 1.  2. The use of the first 180 bp fragment of the polyhedrin gene of claim 1 for protein fusion expression.  An expression vector, which is constructed by sequentially inserting the first 180 bp fragment of the polyhedrin gene and the protease cleavage site fragment of the polyhedrin gene according to the multiple cloning site of the starting vector.  The expression vector according to claim 3, wherein the starting vector is a pET series vector, a cDNA 3 series vector, a pFastBacTM series vector, a pKK series vector or a pBAD series vector.  The expression vector according to claim 4, wherein the departure vector is pET-28a.  The expression vector according to claim 3, wherein the protease cleavage site fragment is a thrombin cleavage site fragment, and the nucleotide sequence thereof is SEQ ID NO: 2.  A fusion protein expression vector, which is characterized in that the first 180 bp fragment of the polyhedrin gene, the protease cleavage site fragment and the target gene fragment of claim 1 are sequentially inserted into the multiple cloning site of the starting vector. .  The fusion protein expression vector according to claim 7, wherein the target group is based on an EGFP gene, an LZM gene, an ORAI gene or an MMgT gene.  A genetically engineered bacterium comprising the fusion protein expression vector of claim 7.  The genetically engineered bacterium according to claim 9, wherein the genetically engineered bacterium is Escherichia coli.