CN106399347A - Preparation method of GyV5 novel annular virus VP3 protein - Google Patents

Abstract

The invention relates to a preparation method of GyV5 novel annular virus VP3 protein. According to the method, pGEX-6p-1 plasmid and GyV5 virus VP3 gene are used as templates, a pGEX-6p-1 linear vector and a GyV5 virus VP3 gene fragment are respectively amplified by PCR, and rapid recombinant cloning is carried out by recombinase Exnase TM II; and positive clone is transformed to colibacillus, so that VP3 protein expression is realized. The VP3 protein can be directly provided as GyV5 diagnosis antigen, and is used as immunogen to obtain an anti-VP3 multi-clone antibody; and an effective immunological reagent is provided for development of GyV5 serological and epidemiological survey and VP3 function research, and domestic and overseas gaps are filled.

Description

A kind of preparation method of GyV5 novel circular virus VP3 protein

technical field

The invention relates to a preparation method of GyV5 novel circular virus VP3 protein. This invention can directly provide VP3 protein as GyV5 diagnostic antigen; obtain anti-VP3 polyclonal antibody as immunogen; provide effective immunological reagents for the development of GyV5 serological epidemiological investigation and VP3 function research, filling the gap at home and abroad.

Background technique

Chicken Infectious Anemia Virus (CAV) has been considered the only member of the genus Gyrovirus in the Circoviridae family. Until 2011, Rijsewijk et al. detected a new type of Circovirus sequence, the second member of the Circovirus genus, named AGV2 from serum samples of infected chickens. In the same year, Sauvage et al. detected the first human circular virus HGyV sequence highly homologous to AGV2 in healthy human skin cotton samples. Since 2012, other novel circoviruses including GyV3, GyV4, GyV5, GyV6, GyV7, GyV8 and GyV9 have been discovered and identified successively, and have potential public health significance. However, there is no serological method to detect GyV5 antigen and its antibody. Therefore, cloning the VP3 gene of the early expression protein of GyV5 virus in vitro, constructing the VP3 expression vector, and realizing its expression will help to carry out in-depth detection of GyV5 protein antigen and its antibody, serological investigation, and clarify the infection of GyV5 in chickens and people. The replication status provides an effective diagnostic reagent; and it is of great significance for exploring the biological function of GyV5VP3. In the construction of traditional expression vectors, it is often necessary to design and select restriction endonuclease sites, and construct vectors by enzyme digestion and ligation to realize the expression of foreign genes. However, sometimes the cloning process is cumbersome and inefficient because no suitable enzyme cutting site can be found. In the present invention, the VP3 gene of GyV5 virus will be cloned by using the recombination enzyme ExnaseTM II in vitro recombination technology independent of enzyme cutting sites and restriction endonucleases, which simplifies the cloning process and realizes rapid cloning and expression of the VP3 gene.

Contents of the invention

The purpose of the present invention is to provide a method for preparing the VP3 protein of the GyV5 novel circular virus to realize the expression of the VP3 protein.

The principle and core key technology of the present invention is to scientifically design the primers for amplifying the pGEX-6p-1 linearized vector and the VP3 gene fragment of GyV5 virus, and to directly VP3 was rapidly recombined and cloned in vitro, transformed into Escherichia coli, induced by IPTG, and expressed in fusion of GyV5 VP3 protein and GST, and purified VP3 fusion protein was obtained.

The preparation method of the GyV5 novel circular virus VP3 protein according to the present invention uses the pGEX-6p-1 plasmid and the GyV5 virus VP3 gene as templates, and PCR amplifies the linearized vector containing pGEX-6p-1 and the GyV5 virus VP3 gene fragment respectively (accompanying drawing 1, step 1), and utilize recombinase ExnaseTM II to carry out rapid recombination cloning (accompanying drawing 1, step 2); Positive clone is transformed into Escherichia coli, realizes VP3 protein expression (accompanying drawing 1, step 3).

The above method comprises the following steps:

1) Using the following primers to amplify the pGEX-6p-1 linearized expression vector by PCR using the pGEX-6p-1 plasmid as a template;

Upstream primer: 5'TAATGACGGTGAAAACCTCTGACACATGC3' (SEQ ID NO.1);

Downstream primer: 5'CATGGGCCCCTGGAACAGAACTTCCAGAT3'' (SEQ ID NO.2);

2) Utilizing the following primers and using the GyV5-VP3 gene as a template, PCR amplifies the VP3 gene;

Upstream primer: 5'GTTCCAGGGGCCCATGTTGGCTGACGAGTT3' (SEQ ID NO.3);

Downstream primer: 5'GTTTTCACCGTCATTAAAGTTCTTCTTGTA3' (SEQ ID NO.4);

3) Rapid recombination and cloning of the PCR products obtained in steps 1) and 2) using the recombinase ExnaseTM II; positive clones were induced by IPTG for expression identification and purification.

The invention discloses PCR amplifying pGEX-6p-1 linearized carrier primers and PCR amplifying the sequences of GyV5 virus VP3 gene primers. The present invention also discloses a direct recombination and cloning technique based on the recombinase ExnaseTM II to directly recombine and clone the linearized pGEX-6p-1 linearized vector and the PCR product of the VP3 gene fragment of GyV5 virus into Escherichia coli. VP3 protein expression.

The primers designed by the present invention and the cloning strategy based on the recombinase ExnaseTM II can rapidly construct the prokaryotic expression vector of the VP3 gene of the GyV5 virus. The GyV5 virus VP3 expression and purified protein obtained by the present invention can directly provide VP3 protein as GyV5 diagnostic antigen; obtain anti-VP3 polyclonal antibody as immunogen; provide effective diagnostic reagents for carrying out GyV5 epidemiological investigation; and further explore VP3 Biological function is of great significance.

Description of drawings

Figure 1 Schematic diagram of the preparation method for the VP3 protein of a new type of GyV5 circular virus

Step 1: Using the artificially synthesized GyV5 virus VP3 gene as a template, use primers and PCR to amplify the pGEX-6p-1 linearized vector and GyV5 virus VP3 gene fragments respectively.

Step 2: Rapid recombination and cloning using the recombinase ExnaseTM II.

Step 3: Positive clones were transformed into E. coli to achieve VP3 protein expression.

Figure 2 PCR amplification of VP3 fragment of GyV5 virus

Lane 1, PCR product of VP3 fragment of GyV5 virus; Lane M, DNA Marker.

Figure 3 PCR amplification of linearized vector pGEX-6p-1

Lane 1, PCR product of linearized vector pGEX-6p-1; lane M, DNA Marker.

Figure 4 SDS-PAGE analysis of the expression of VP3 gene of GyV5 virus

Lanes 1 and 2 represent supernatants and precipitates of VP3 sonicated samples induced by IPTG; lanes 3 and 4 are purified VP3 proteins; lanes 5 and 6 are supernatants and precipitates of GST sonicated samples.

Figure 5 Western blot identification of anti-AGV5VP3 protein polyclonal antibody

1, 293T cell lysate transfected with EGFP-VP3 expression plasmid; 2, 293T cell lysate transfected with control EGFP expression plasmid.

detailed description

In order to better understand the content of the present invention, the following embodiments give an example of the preparation of the VP3 protein of the GyV5 novel orbivirus in conjunction with the accompanying drawings.

Example

1) Design and amplify the primers containing the pGEX-6p-1 linearized vector and the VP3 gene fragment of the GyV5 virus: the upstream primer for amplifying the pGEX-6p-1 linearized vector is located at position 1022-1047 of the pGEX-6p-1 plasmid; and at the 5' There is an extra TAA base at the end; the downstream primer for amplifying the pGEX-6p-1 linearization vector is located at position 916-941 of the pGEX-6p-1 plasmid; and there is an extra CAT base at the 5' end. Amplify GyV5 virus VP3 gene upstream primers include VP3 gene start codon ATG and the following 14 bases, and have 16 reverse complementary bases with pGEX-6p-1 linearized vector downstream primers at the 5' end; The downstream primer of VP3 gene of GyV5 virus includes VP3 gene stop codon TAA and its first 14 bases, and has 16 reverse complementary bases at the 5' end with the upstream primer of pGEX-6p-1 linearized vector. The specific primer sequences are as follows, synthesized by Life Technologies Shanghai Thermo Fisher Scientific Co., Ltd.

Primers for pGEX-6p-1 linearized expression vector:

Upstream primer: 5'TAATGACGGTGAAAACCTCTGACACATGC3' (SEQ ID NO.1);

Downstream primer: 5'CATGGGCCCCTGGAACAGAACTTCCAGAT3'' (SEQ ID NO.2);

GyV5-VP3 gene primers:

Upstream primer: 5'GTTCCAGGGGCCCATGTTGGCTGACGAGTT3' (SEQ ID NO.3);

Downstream primer: 5'GTTTTCACCGTCATTAAAGTTCTTCTTGTA3' (SEQ ID NO.4).

2) PCR amplification of the pGEX-6p-1 linearized vector and the VP3 gene fragment of the GyV5 virus: the pGEX-6p-1 plasmid and the synthetic GyV5VP3 gene (GenBank: KF294861.1) were used as templates, and the primers in the above 1) were used as primers PCR amplification. Step 1 in Figure 1. The PCR amplification reaction system is: 40 μl water, 5 μl 10 times buffer, 1 μl 10 mM dNTP, 1 μl 10 μmol upstream primer, 1 μl 10 μmol downstream primer, 1 μl 10 ng/μl pGEX-6p-1 plasmid or pcGyV5-VP3 plasmid, 1 μl commercial Phanta Super-Fidelity DNA Polymerase. The cycle parameters of the PCR amplification reaction were: denaturation at 94°C for 5 minutes, followed by 30 cycles (denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 3 minutes), and finally extension at 72°C for 10 minutes. After PCR, the PCR products were electrophoresed on 1% agarose gel. As shown in Figure 2, where lane M is the DNA Marker, where lane 1 is the PCR product of the VP3 fragment of the GyV5 virus. As shown in Figure 3, lane M is the DNA Marker, and lane 1 is the PCR product of the linearized vector pGEX-6p-1.

3) Rapid cloning of the VP3 fragment of GyV5 virus into the pGEX-6p-1 vector: the above purified expression linearized vector pGEX-6p-1 and the PCR product of the VP3 fragment of GyV5 virus were recombined and cloned under the action of commercially available recombinase ExnaseTM II. Step 2 in Figure 1. The specific recombination reaction system is as follows: 50-100ng of the purified GyV5 virus VP3 fragment product, 50ng of the purified pGEX-6p-1 expression linearization vector, 2μl of commercially available ExnaseTM II enzyme, 4μl of 5-fold buffer, and the rest added water to 20μl . After reacting at 37°C for 30 minutes, the reaction was placed on ice for 5 minutes. Then 20 μl of the reaction was transformed into conventional competent bacteria and coated with LB plates. Bacterial clones were picked for plasmid preparation the next day, and positive clones were identified.

4) Induced expression of GyV5 virus VP3 protein and its purification: the obtained positive clone containing GyV5 virus VP3 gene (named pGEX-VP3) was transformed into BL21 bacteria, and the bacteria were collected after IPTG induction (0.1mmol/ml), and ultrasonic ( 40 Hz) broken. After the sonicated sample was centrifuged, the supernatant and the precipitate were separated for SDS-PAGE (5% stacking gel, 10% separating gel) and Western blot analysis (using anti-mouse GST monoclonal antibody as the primary antibody, goat anti-mouse HRP labeled IgG is the secondary antibody) to identify the expression. In Figure 4, VP3 protein can be present in soluble form in the supernatant of sonicated samples. On the basis of confirming the soluble expression of VP3, the VP3 protein was purified by passing the supernatant of the sonicated sample through a GST purification column. Lanes 1 and 2 in Figure 4 represent the supernatant and precipitate of the ultrasonically lysed sample before IPTG-induced VP3 purification, lanes 3 and 4 are the SDS-PAGE analysis results after VP3 protein purification, and lanes 5 and 6 are the supernatant and precipitate of the GST ultrasonically lysed sample. In order to further determine the antigenicity of the purified protein and evaluate whether it can be used as an immunogen and a diagnostic antigen, the purified VP3 protein was immunized into mice, and the obtained mouse polyclonal antibody against GyV5 virus VP3 protein was proved by western blot analysis. It can specifically react with the VP3 protein expressed in 293T cells (Figure 5). This result shows that the VP3 protein expressed in the present invention has good reactivity and immunogenicity, and will have a good application prospect in GyV5 serological diagnosis.

Claims (2)

1. a kind of preparation method of GyV5 New Ring-like Type virus VP 3 albumen it is characterised in that with pGEX-6p-1 plasmid and GyV5 virus VP 3 gene is template, and PCR amplifies linearized vector containing pGEX-6p-1 and GyV5 virus VP 3 gene piece respectively Section, and carry out Quick Casting clone using recombinase ExnaseTM II；Positive colony is transformed into Escherichia coli, realizes VP3 albumen Expression.

2. method according to claim 1 is it is characterised in that comprise the following steps：

1) utilize following primer, with pGEX-6p-1 plasmid as template, PCR amplifies pGEX-6p-1 linearisation expression vector；

Upstream primer：5‘TAATGACGGTGAAAACCTCTGACACATGC3’；

Downstream primer：5‘CATGGGCCCCTGGAACAGAACTTCCAGAT3’；

2) utilize following primer, with GyV5-VP3 gene as template, PCR amplifies VP3 gene；

Upstream primer：5‘GTTCCAGGGGCCCATGTTGGCTGACGAGTT3’；

Downstream primer：5‘GTTTTCACCGTCATTAAAGTTCTTCTTGTA3’；

3) utilize recombinase ExnaseTM II to step 1) and 2) PCR primer that obtains carries out Quick Casting clone；Positive colony Carry out expression identification and purifying after IPTG induction.