CN106565830A - Construction of Newcastle disease virus HN protein 119-site amino acid mutant - Google Patents

Abstract

The invention belongs to the field of protein engineering, in particular to the construction of the 119-position amino acid mutant of Newcastle disease virus HN protein. A mutant of the HN protein of Newcastle disease virus is characterized in that the mutant adopts the method of site-directed mutation and uses Newcastle disease virus as a template, and the 119th amino acid of the glycosylation site of HN protein is site-directedly mutated from AAT to CAG, and passed through the large intestine Bacillus competent cell HST08 was expressed, the positive plasmid was extracted, and the 119-position amino acid mutant of HN protein of Newcastle disease virus was obtained. The study of this mutant is helpful to study the effect of the mutation of the glycosylation site on the expression of the virus, the spatial structure of the protein, and the activity of the virus, and can determine whether the replication and pathogenicity of the virus have changed.

Description

Construction of 119-amino acid mutant of HN protein of Newcastle disease virus

technical field

The invention belongs to the field of protein engineering, in particular to the construction of the 119-position amino acid mutant of Newcastle disease virus HN protein.

Background technique

The construction technology of in vitro mutants is a powerful tool for studying protein structure and function, and it is also a common method for us to modify and optimize genes in the laboratory. The structure of a protein determines its function, and the relationship between the two is one of the key directions of current proteome research. The targeted change, deletion or insertion of a specific base of a known gene can change the corresponding amino acid sequence, thereby affecting the structure of the protein. A series of studies on the expression products of the mutated gene will help to understand the specific region of the protein function, to investigate the structure/domain of a protein. Usually, overlap extension method, primer method, one-step PCR method and other methods are used to achieve targeted deletion, insertion or base substitution of the target gene. Therefore, site-directed mutagenesis in vitro is a powerful tool for gene function research. The post-translational modification of multiple sites within proteins plays an important role in the regulation of gene function. The point mutants of genes play a very critical role in the study of their structure and function. Therefore, efficient, rapid and accurate construction of single or multiple point mutants of genes is of great significance in the study of gene functions.

Newcastle disease is an acute and highly contagious poultry disease caused by Newcastle Disease Virus (NDV), which mainly affects chickens and turkeys. It is recognized as one of the two most important poultry infectious diseases in the world. The pathogenicity of NDV is mainly achieved through the joint action of its functional protein-fusion protein F and hemagglutinin neuraminidase protein HN, and glycosylation, as a major post-translational modification, has a significant impact on the function of the protein. Significant influence. Therefore, mutations in glycosylation sites can affect the synthesis of viral proteins, thereby affecting their activity and function. Past studies often focused on the glycosylation site of F protein, while ignoring the important role of the glycosylation site of HN protein in recognizing sialic acid receptors and promoting cell fusion. Therefore, the research on the glycosylation site of HN protein It is of great significance to study the effect of glycosylation site mutation on virus expression, protein spatial structure, virus activity, and whether the replication and pathogenicity of the virus are changed.

Contents of the invention

In order to overcome the defects in the prior art, the present invention provides a 119-position amino acid mutant of the Newcastle disease virus HN protein, and the study of the mutant helps to study the effect of the mutation of the glycosylation site on the expression of the virus and the spatial structure of the protein. , the impact of virus activity, and can determine whether the replication and pathogenicity of the virus have changed.

The present invention also provides a method for constructing the 119-position amino acid mutant of the Newcastle disease virus HN protein, which has high technical requirements.

A mutant of the HN protein of Newcastle disease virus, the mutant uses the cDNA of Newcastle disease virus as a template by site-directed mutation, and the 119th amino acid of the glycosylation site of the HN protein of the Newcastle disease virus is site-directedly mutated by AAT It is CAG, which is expressed by Escherichia coli competent cell HST08, and the positive plasmid is extracted, and after verification, the 119-amino acid mutant of the HN protein of Newcastle disease virus is obtained.

The construction of the above-mentioned mutant mainly includes the following steps:

(1) Carry out fixed-point transformation on the glycosylation site of the HN protein of Newcastle disease virus. Using the cDNA of Newcastle disease virus as a template, two pairs of specific primers were used for PCR amplification to obtain two sets of amplified products. The product was subjected to electrophoresis and gel cutting to recover the target gene fragments NDV-1 Insert A and NDV-1 Insert B;

(2) Digest the constructed plasmid pVAX containing the glycosylation site of HN protein with restriction endonuclease Sse8387I, and purify the digested product to obtain pVAX-NDV-Sse8387I;

(3) Digest the pVAX-Sse8387I obtained in step (2) with the restriction endonuclease SfiI to obtain pVAX-SfiI/Sse8387I, and perform electrophoresis to cut the pVAX-SfiI/Sse8387I to obtain NDV-Vector;

(4) Ligate the NDV-1 Insert A and NDV-1 Insert B obtained in step (1) with the NDV-Vector obtained in step (3), transform the ligated product into Escherichia coli HST08 competent cells, and transform the transformed cells Spread it on a plate, extract the plasmid for identification, screen the positive strain, and name it NDV-1-5, which is the 119-position amino acid mutant of the Newcastle disease virus HN protein.

Further, the nucleotides of the specific primers used for the gene fragment NDV-1 Insert A described in step (1) are: SEQ ID NO.1 and SEQ ID NO.2; the gene fragment NDV described in step (1) -1 The nucleotides of the specific primers used for Insert B are: SEQ ID NO.3 and SEQ ID NO.4.

Further, the reaction conditions of the PCR reaction in step (1) are: 98°C for 10 sec, 55°C for 15 sec, 72°C for 120 sec, a total of 30 cycles; then 72°C for 10 min.

Further, the enzyme digestion reaction conditions in step (2) are: reaction at 37°C for 2 hours; the enzyme digestion reaction conditions in step (3) are: reaction at 50°C for 4 hours; the ligation reaction in step (4) The condition is 50°C for 15 minutes.

Beneficial effect

(1) In this study, a single point mutant of amino acid 119 of the glycosylation site of Newcastle disease virus HN protein has been constructed, that is, the AAT base at the 355th-357th bp is mutated to CAG. The obtained NDV infectious clone with a single point mutation in the glycosylation site is helpful to study the influence of the mutation in the glycosylation site on the expression of the virus, the spatial structure of the protein, and the activity of the virus, and can also determine the replication and sex of the virus. Whether the pathogenicity has changed.

(2) After the mutants of the present invention are successfully constructed and rescued, the effect of the mutation of the 119 glycosylation site of the HN protein on the virus reinfection ability can be clarified. On the basis of constructing mutant infectious clone HN119 and successfully rescuing the virus, the change of virus infectivity was determined by TCID ₅₀ . The results showed that the first three generations of rescued viruses multiplied slowly, and the virus multiplication accelerated from the fourth generation, and the virus valence of the fifth and sixth generations was basically stable and consistent. The virulence of the mutant virus is significantly lower than that of the non-mutant virus; therefore, the mutation of the HN119 glycosylation site can reduce the infectivity of the virus. Therefore, it can be judged that the 119 amino acid glycosylation site has an important role in the pathogenicity of the virus. Play a role.

(3) Quantitative detection of virus replication in cells was carried out on the mutant virus and the original virus by real-time fluorescence quantitative PCR with probes. Compared with the parental virus, the mutant virus was different, and the viral load was increased. The detection results showed that the mutation of the HN119 glycosylation site could enhance the replication ability of the virus in vitro.

Description of drawings

Fig. 1 electrophoresis diagram of PCR amplification product of Newcastle disease virus;

Fig. 2 Electropherogram of pVAX-SfiⅠ/Sse8387Ⅰ.

detailed description

The present invention will be further described in conjunction with the examples. It should be noted that the following descriptions are only for explaining the present invention and not limiting its content.

Example 1

1. Cloning of full-length Newcastle disease virus gene

(1) Design of primers for full-length cloning of Newcastle disease virus

The sequences of the amplification primers are listed in Table 1:

Table 1

(2) Full-length PCR amplification of Newcastle disease virus

Using the cDNA of Newcastle disease virus as a template, the above two pairs of primers and PrimerSTAR® HS (Premix) high-fidelity enzyme were used for PCR amplification. The reaction system is as follows:

Component Reaction volume

NDV cDNA (10ng/ul) 1 ul

Primer ^*1 (20pmol/ul) 0.5ul

Primer ^*2 (20pmol/ul) 0.5ul

PrimerSTAR HS (Premix) 25ul

dH ₂ O Upto50ul

The reaction conditions are: 98°C for 10 sec, 55°C for 15 sec, 72°C for 120 sec, a total of 30 cycles; then 72°C for 10 min.

2. PCR product electrophoresis and gel cutting recovery

Take 5 ul of the PCR product obtained in step 1 and carry out 1% agarose gel electrophoresis detection, as shown in Figure 1.

Among them, the length of amplified fragment 1 is about 500bp, and the length of amplified fragment 2 is about 2000bp, all of which are in line with the expected amplified length.

Use TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 for gel cutting and recovery. The names of the amplification products corresponding to the amplification primers and the recovered products after gel cutting are shown in Table 2 below:

Table 2

3. Digestion of plasmid pVAX and product recovery

Plasmid pVAX was digested with Sse8387I and SfiI respectively. Sse8387I and SfiI enzymes were respectively purchased from Dalian TakaRA Company. The Sse8387I enzyme digestion system was as follows:

Component Reaction volume

pVAX (50ng/ul) 30ul

10× Quick Cut Buffer 5ul

Sse8387Ⅰ（10U/ul） 2ul

dH ₂ O Up to 50ul

The reaction conditions were 2 hours of digestion at 37°C. After the reaction, the digested product was purified by TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 and named pVAX-Sse8387Ⅰ;

pVAX-Sse8387Ⅰ was digested with SfiⅠ, and the SfiⅠ digestion system was as follows:

Component Reaction volume

pVAX-Sse8387Ⅰ 43ul

10×M Buffer 5ul

SfiⅠ（10U/ul） 2ul

Total 50ul

The enzyme digestion reaction condition is 50°C for 4 hours. After the reaction, take 5ul of the digested product pVAX-SfiⅠ/Sse8387Ⅰ, and perform 1% agarose gel electrophoresis for detection. After the 16 kbp DNA fragment, named NDV-Vector.

4. Ligation of the vector NDV-Vector and the target gene fragment

Using the In-Fusion HD Cloning Kit, perform an In-Fusion reaction on the target gene fragments NDV-1 Insert A and NDV-1 Insert B prepared in step 2 and the NDV-Vector prepared in step 3. The reaction system is as follows:

Component Reaction volume

NDV-Vector (50ng/ul) 2ul

NDV-1 Insert A (50 ng/ul) 1 ul

NDV-1 Insert B (50 ng/ul) 1 ul

5xIn-Fusion HD Enzyme Premix 2 ul

dH ₂ O Up to 10 ul

The reaction condition was 50°C for 15 minutes.

5. Colony culture

After the ligation reaction was completed, the product was directly transformed into competent cells. The competent cells were selected from Escherichia coli HST08 strain, which was purchased from Dalian TakaRA Company, and cultured at 37°C for 12 hours. The positive colonies were selected for planting, and the extracted plasmid was named NDV-1-5.

6. Sequencing identification

The NDV-1-5 plasmid was sequenced using primers CTG0450 P10 and CTG0450 P12, and the nucleotide sequences of CTG0450 P10 and CTG0450 P12 are shown in Table 3 below:

table 3

Example 2

The change in infectivity of the mutant virus was determined _by TCID50 (Cell lethal dose). After the cultured cells were routinely digested with 0.25%-EDTA trypsin, they were added to 96-well plates, 100 μL per well, and the cell cultures of the rescued virus NDV-1-5 were inoculated at a dilution of ^10-1 , and placed at 37 ° C, 5% In an incubator with CO ₂ ; after 12 hours, add 10 μL D-glucosamine to each well for 15 minutes, then wash with 2% DMEM medium, add 200 μL 2% DMEM medium to each well after washing, and continue to cultivate; 48 hours after treatment , discard the maintenance solution, wash 3 times with 100 μL of PBS per well, add 4% paraformaldehyde after washing and fix at room temperature for 45min-60min; remove the fixative, wash 3 times with PBS, use NDV HN monoclonal antibody as the primary antibody, Dilute at 1:200, add 100 μL to each well, incubate at room temperature for 1 h in the dark; wash with PBS three times, then add FITC-labeled goat anti-mouse fluorescent secondary antibody, dilute at 1:800, incubate at room temperature for 1 h. After washing with PBS three times, observe under the microscope; calculate its TCID ₅₀ according to the Reed-Muench method. Data analysis showed that the virulence of the HN119 amino acid mutant virus was Lg3.1, which was significantly lower than the TCID ₅₀ value of Lg3.9 of the non-mutant virus. Since the virulence of the mutant virus is significantly lower than that of the non-mutant virus; therefore, the mutation of the HN119 glycosylation site can reduce the infectivity of the virus. Therefore, it can be judged that the 119 amino acid glycosylation site has an important role in the pathogenicity of the virus. play a role.

Quantitative real-time PCR reaction to measure the replicability changes of mutant viruses

The reaction system (25 μL) is as follows: Buffer 5 μL, mixed enzyme 2 μL, probe mixed primer (10 μmol/L) 1 μL, viral RNA template 2 μL, sterilized water 15 μL. The reaction conditions of fluorescent quantitative PCR were: 50°C for 30 min, 95°C for 5 min; followed by 40 cycles of 95°C for 20 s, 52°C for 20 s, and 70°C for 25 s. The virus content was judged by the fluorescence standard curve. The analysis of the results showed that the mutant virus was different from the parental virus, and the viral load was increased. This shows that the mutation of the HN119 glycosylation site can enhance the replication ability of the virus in vitro.

From the above, it can be known that point mutants of genes play a very critical role in the study of their structure and function. Therefore, efficient, rapid and accurate construction of single or multiple point mutants of genes is of great significance in the study of gene functions. In this study, a single-point mutant of the 119th amino acid of the glycosylation site of HN protein was constructed, that is, the AAT base at the 355th-357th bp was mutated to CAG. The difference in infectivity between the obtained glycosylation site single-point mutant and the unmutated original virus provides an important basis for further research on the function and pathogenic mechanism of viral proteins.

<110> Institute of Poultry, Shandong Academy of Agricultural Sciences

<120> Construction of 119-position amino acid mutant of Newcastle disease virus HN protein

<160> 7

<210> 1

<211> 3152

<212>DNA

<213> Artificial sequence

ccggatttgg gtgatcaaag ggcaacatac gggtagaacg gccagagagg ccactcctta 60

gccaggaatc gggcctcaca ccatccgttc taccgcatca ccaatagcgg tttttagtca 120

tggaccgtgt agttagccaa gttgcgctag agaacgatga aagagaggcg aaaaatacat 180

ggcgcttggt atttcggacc gcagtcttac ttttaatagt agtgaccttt tccatctctg 240

ctgccgccct gatgtacagt atggaggcta gcacacctgg tgaccttgta ggcatactga 300

ctgcgatctc cagggcagaa gaaaagatta catctgcact cggttccaat caagatgtag 360

tagataggat atataagcag gtggccctcg aatctccgtt ggcattgctc aacaccgaat 420

ctataattat gagtgcaata acgtccctct cttaccagat caatggagct gcacagaaca 480

gtgggtgtgg ggcacctgtt catgacccgg attatatcgg agggataggt aaagaactca 540

ttgtggatga tgctagtgat gtcacatcat tctatccctc tgcgttccaa gaacacctga 600

attttatccc ggcgcccact acaggatcag gttgcactcg gataccctca ttcgacatga 660

gtgctaccca ctactgttac actcataatg tgatattgtc tggctgcaga gatcactcac 720

actcacatca gtatttggca cttggtgtgc ttcggacatc tgcaacaggg agggtattct 780

tttctactct gcgttccatc aatttggatg acaaccaaaa tcggaagtct tgcagtgtga 840

gtgcaactcc cttaggttgc gatatgttgt gctctaaagt cacggaaact gaggaagaag 900

attataattc agttatcccc acaccaatgg tacatggggag gctggggttt gacggccaat 960

accatgagaa ggacctggat gtcgcaacat tatttgggga ctgggtggca aattaccctg 1020

gggtgggagg agggtctttt attgacaacc gcgtatggtt cccagtctat ggagggctaa 1080

aacccaattc gcctagtgac actgcacaag aggggagata tgtaatatac aagcggtaca 1140

atgacacatg cccagatgag caagactacc agattcggat ggctaagtct tcatataagc 1200

ctgggcggtt tggtgggaaa cgcgtacagc aggccatcct atccatcaag gtatcaacat 1260

ccttgggtga ggacccggtg ctgactgtac cgcccaacac aatcacactt atgggggccg 1320

aaggcagagt tctcacagta gggacatctc atttctttta ccagcgaggg tcatcatact 1380

tctctcccgc cttattatac cctatgacag tcgacaataa aacagccact cttcatagtc 1440

cttatgcatt caatgctttc actcggccag gtagtgtccc ttgccaggct tcagccagat 1500

gccctaactc gtgtgttact gaggtctaca ctgatccata ccccttagtc ttccatagga 1560

accacacttt gcgaggggta ttcgggacaa tgcttgatga taaacaagca agactcaacc 1620

ctgtatctgc agtatttgat aacatatctc gcagtcgcat aactcgggtg agttcaagca 1680

gtaccaaggc agcataacacg acatcaacat gttttaaagt tgtcaagacc aataaaacct 1740

attgcctcag cattgcagaa atatccaata ccctcttcgg ggaattcagg attgtccctt 1800

tattagtcga gattctcaag gatgatggga tttaagaagc caggtctggc tggttgagcc 1860

agctgtgaaa gggccgggaa gatgacactg cgccacccat cctttgtagc accaggaatc 1920

aaactgagaa ccggcacagg ctcaaatcat acgctgccgg tcagccacaa tcagctatcg 1980

ccaatgcgat tagtctggat cttgccaata gtcacttgat taagaaaaat tatagatggt 2040

agtgagatac gagagaaagc aactcacggt ggataacacg ggtaggacat ggcgagctcc 2100

ggtcccgaga gggcagagca ccagattatc ctaccagagt cacatctgtc ttcaccatg 2160

gtcaagcaca aactgctcta ttactggaaa ttaacagggc taccacttcc tgacgaatgc 2220

gacttcgacc accttattat cagtcgacaa tggaagaaag tacttgaatc ggccactcct 2280

gacattgaga gaatgataaa actcgggcgg tcagtacacc agactctcaa ccacaattcc 2340

aggataactg gagtactaca tcccagatgt ttagaagaat tggctagtat tgaggtccct 2400

gattcaacca acaaatttcg gaagatcgaa aagaagatcc agattcacaa cacaaggtat 2460

ggagaactat tcactcggct gtgcacgcat gtagaaaaga aattattggg gtcgtcttgg 2520

tctagcaatg tcccacgatc agaggaattc agcagcatcc gtacagatcc ggcattctgg 2580

tttcattcaa aatggtccac ggccaagttt gcgtggctcc atataaaaca ggtccaaagg 2640

catctaattg tagcagcaag aacaagatcc gcagtcaaca aattagtaac actgacccat 2700

aaggtaggcc aagtatttgt tactcctgag cttgttattg tgacacatac agatgagaac 2760

aagttcacgt gtcttaccca ggaacttgtg ttgatgtatg cagatatgat ggagggcaga 2820

gatatggtca acataatatc atccacggca gcacatctta ggagcttatc agagaaaatt 2880

gatgacattc tgcggttgat agatgctttg gcaaaagaat tgggcaatca ggtctacgat 2940

gttgtagcac taatggaggg attcgcatac ggcgctgttc agctgcttga gccgtcaggt 3000

acatttgcag gggatttctt tgccttcaac ctgcaggagc tcaaagatac tataatcgga 3060

ctcctcccca aggatatcac agaatctgtg actcatgcaa tcgccaccgt attctctggc 3120

ttagaacaaa atcaagcagc cgagatgttg tg 3152

<160> 7

<210> 2

<211> 25

<212>DNA

<213> Artificial sequence

cgggtagaac ggccagagag gccac 25

<160> 7

<210> 3

<211> 25

<212>DNA

<213> Artificial sequence

tggagctgca cagaacagtg ggtgt 25

<160> 7

<210> 4

<211> 21

<212>DNA

<213> Artificial sequence

gtatctttga gctcctgcag g 21

<160> 7

<210> 5

<211> 23

<212>DNA

<213> Artificial sequence

cactgttctg tgcagctcca ttg 23

<160> 7

<210> 6

<211> 20

<212>DNA

<213> Artificial sequence

gcagaggtat ctccaagagc 20

<160> 7

<210> 7

<211> 20

<212>DNA

<213> Artificial sequence

atgttgcgac atccaggtcc 20

Claims (5)

1. a kind of newcastle disease virus HN protein mutant, it is characterised in that the cDNA with Avian pneumo-encephalitis virus as template, by the new city The amino acids of HN protein glycosylations site the 119th of epidemic disease poison are CAG by rite-directed mutagenesises by AAT, and Jing E. coli competents are thin Born of the same parents HST08 strains are expressed, and extract positive plasmid, and after checking is correct the amino acids mutant of newcastle disease virus HN protein 11 9 is obtained.

2. a kind of newcastle disease virus HN protein mutant described in claim 1, it is characterised in that the structure master of the mutant Comprise the following steps：

（1）Fixed point transformation is carried out with the cDNA of Avian pneumo-encephalitis virus as template to the HN protein glycosylations site of Avian pneumo-encephalitis virus, is used Two pairs of specific primers enter performing PCR amplification, obtain two groups of amplified productions, carry out electrophoresis to two groups of PCR primers respectively and cut glue reclaim Genes of interest fragment NDV-1 Insert A and NDV-1 Insert B；

（2）The plasmid pVAX containing HN protein glycosylations site to building carries out enzyme action with restricted enzyme Sse8387 I Afterwards, purification is carried out to digestion products, obtains pVAX-Sse8387 I；

（3）To step（2）The pVAX-Sse8387 I for obtaining restricted enzyme Sfi I carry out after enzyme action, obtaining pVAX-Sfi I/Sse8387 I, carries out electrophoresis and to cut obtain after glue NDV-Vector to I/Sse8387 of pVAX-Sfi I；

（4）By step（1）The NDV-1 Insert A and NDV-1 Insert B for obtaining and step（3）The NDV-Vector for obtaining It is attached, connection product is transformed in escherichia coli HST08 competent cells, transformed cells are coated on flat board, carries Plasmid identification is taken, positive strain is screened, NDV-1-5, as the amino acids mutant of newcastle disease virus HN protein 11 9 is named as.

3. preparation method according to claim 2, it is characterised in that step（1）Described genetic fragment NDV-1 The nucleotide of the specific primer used by Insert A is：SEQ ID NO.1 and SEQ ID NO.2；Step（1）Described gene The nucleotide of the specific primer used by fragment NDV-1 Insert B is：SEQ ID NO.3 and SEQ ID NO.4.

4. preparation method according to claim 2, it is characterised in that step（1）The reaction condition of described PCR reactions For：98 DEG C of 10sec, 55 DEG C of 15sec, 72 DEG C of 120sec, totally 30 circulations；Then 72 DEG C of 10min.

5. preparation method according to claim 2, it is characterised in that step（2）Described endonuclease reaction condition is：37℃ Reaction 2 hours；Step（3）Described endonuclease reaction condition is：50 DEG C are reacted 4 hours；Step（4）The bar of described coupled reaction Part is 50 DEG C of reaction 15min.