CN106349348A - Preparation method of GyV7 ring virus VP3 protein - Google Patents

Abstract

The invention provides a preparation method of GyV7 circular virus VP3 protein. The method is based on the recombinase ExnaseTM II to directly recombine and clone the linearized pGEX-6p-1 vector and the PCR product of the VP3 gene fragment of GyV7 virus without enzyme digestion and ligation; and transform it into Escherichia coli to realize the expression of VP3 protein; wherein, The nucleotide sequences of the upstream and downstream primers used for PCR amplification of the VP3 gene of GyV7 virus are respectively shown in SEQ ID NO.3 and SEQ ID NO.4. The method is simple, fast and efficient, and the obtained GyV7 virus VP3 expressed and purified protein can directly provide VP3 protein as GyV7 diagnostic antigen; obtain anti-VP3 polyclonal antibody as immunogen; provide effective diagnostic reagents for GyV7 epidemiological investigation ; And it is of great significance to further explore the biological function of VP3.

Description

A kind of preparation method of GyV7 circular virus VP3 protein

technical field

The invention relates to a preparation method of GyV7 circular virus VP3 protein.

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. (Rijsewijk FA, Dos Santos HF, Teixeira TF, Cibulski SP, Varela AP, Dezen D, etal. Discovery of a genome of a distant relative of chicken anemia virus reveals a new member of the genus Gyrovirus. Archives of virology.2011Jun; 156(6):1097-100), a new type of circovirus sequence was detected from the serum samples of diseased chickens, that is, the second member of the circovirus genus, named AGV2. In the same year, Sauvage et al. (Sauvage V, Cheval J, Foulongne V, Gouilh MA, Pariente K, Manuguerra JC, et al. Identification of the first human gyrovirus, a virus related to chicken anemia virus. Journal of virology. 2011Aug; 85(15): 7948-50) detected the first human orbivirus HGyV sequence highly homologous to AGV2 in skin cotton samples of healthy people. 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 currently no serological method for the detection of GyV7 antigen and its antibody. Therefore, cloning the early expression protein VP3 gene of GyV7 virus in vitro, constructing VP3 expression vectors, and realizing its expression will help in-depth detection of GyV7 protein antigen and its antibody, serological investigation, and clear infection of GyV7 in chickens and people. The replication status provides an effective diagnostic reagent; and it is of great significance for exploring the biological function of GyV7VP3. 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.

Contents of the invention

The purpose of the present invention is to provide a simple, efficient and fast preparation method of GyV7 circular virus VP3 protein expression vector, and realize the expression of VP3 protein. The principle and the core key technology of the present invention are: scientifically design the primers for amplifying the pGEX-6p-1 linearized vector and the VP3 gene fragment of GyV7 virus, and use the commercially available recombinase ExnaseTM II for the ligation reaction without enzyme digestion. VP3 was rapidly recombined and cloned directly in vitro, transformed into Escherichia coli, induced and expressed by IPTG, and the fusion expression of VP3 protein of GyV7 and GST was realized, and purified VP3 fusion protein was obtained.

In order to achieve the above object, the technical solution of the present invention is specifically as follows:

The invention provides a primer for PCR amplification of the pGEX-6p-1 linearized vector, the nucleotide sequence of which is:

Upstream primer 1: 5'-TAATGACGGTGAAAACCTCTGACACATGC-3'; (SEQ ID NO.1)

Downstream primer 1: 5'-CATGGGCCCCTGGAACAGAACTTCCAGAT-3'. (SEQ ID NO.2)

The upstream primer for amplifying the pGEX-6p-1 linearization vector is located at positions 1022-1047 of the pGEX-6p-1 plasmid; and has an extra TAA base at the 5' end; the downstream primer for amplifying the pGEX-6p-1 linearization vector is located in pGEX -Position 916-941 of the 6p-1 plasmid; and has an extra CAT base at the 5' end.

The invention provides a primer for PCR amplification of the GyV7 virus VP3 gene, the nucleotide sequence of which is:

Upstream primer 2: 5'-GTTCCAGGGGCCCATGGCACACCTATACAC-3'; (SEQ ID NO.3)

Downstream primer 2: 5'-GTTTTCACCGTCATTACAGTCTTGCGGCGT-3'. (SEQ ID NO.4)

The upstream primers for amplifying the VP3 gene of GyV7 virus include the VP3 gene start codon ATG and the following 14 bases, and there are 16 reverse complementary bases at the 5' end with the downstream primer of the pGEX-6p-1 linearized vector; The downstream primer of VP3 gene of GyV7 virus includes VP3 gene termination 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 present invention also provides a method for preparing the VP3 protein of GyV7 circular virus. The method is based on the recombinase ExnaseTM II to directly recombine and clone the linearized pGEX-6p-1 vector and the PCR product of the VP3 gene fragment of GyV7 virus without enzymatic digestion. technology; and transformed into Escherichia coli to realize VP3 protein expression; wherein, the primers for PCR amplification of GyV7 virus VP3 gene are divided into upper and lower primers, and the nucleotide sequence of the upper primers is as shown in SEQ ID NO.3 , the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

It specifically includes the following steps:

1) PCR amplification of the pGEX-6p-1 linearized vector and the GyV7 virus VP3 gene fragment: using the pGEX-6p-1 plasmid and the GyV7 virus VP3 gene as templates, using the above primers, PCR amplified the fragments containing pGEX-6p-1 respectively Linearized vector and GyV7 virus VP3 gene fragment (accompanying drawing 1, step 1);

2) Quickly clone the GyV7 virus VP3 fragment into the pGEX-6p-1 vector: utilize the recombinase ExnaseTM II to carry out rapid recombination cloning (accompanying drawing 1, step 2);

3) The positive clones were transformed into Escherichia coli to realize the expression of VP3 protein (accompanying drawing 1, step 3).

The present invention also provides the above-mentioned GyV7 circular virus VP3 protein preparation method, and the application of the above-mentioned primers in the preparation of GyV7 diagnostic antigen, anti-VP3 polyclonal antibody and the preparation of GyV7 epidemiologically effective diagnostic reagents.

Technical scheme of the present invention has reached following beneficial effect:

1) In the present invention, the VP3 gene of the GyV7 virus is cloned using the recombinase ExnaseTM II in vitro recombination technology independent of restriction sites and restriction endonucleases, which simplifies the cloning process and realizes the rapid cloning and expression of the VP3 gene;

2) The primers designed by the invention and the cloning strategy based on the recombinase ExnaseTM II can rapidly construct the prokaryotic expression vector of the VP3 gene of the GyV7 virus.

3) the GyV7 virus VP3 expression and purified protein obtained by the present invention can directly provide VP3 protein as GyV7 diagnostic antigen; obtain anti-VP3 polyclonal antibody as immunogen; provide effective diagnostic reagent for carrying out GyV7 epidemiological investigation; and provide further It is of great significance to explore the biological function of VP3.

4) The present invention will obtain GyV7 circular virus VP3 protein expression vector, realize the expression of VP3 gene in Escherichia coli, and obtain purified VP3 fusion protein. The VP3 fusion protein constructed and purified by this expression vector will provide an effective immunological reagent for the establishment of a serological diagnostic method for detecting GyV7 antigen and antibody, and to explore the biological function of VP3, filling the gap at home and abroad. Therefore, the present invention has certain application value.

Description of drawings

Fig. 1 is a kind of GyV7 circular virus VP3 protein preparation method strategy. Step 1: Using the artificially synthesized GyV7 virus VP3 gene as a template, using the primers in Table 1, PCR amplifies the fragments containing the pGEX-6p-1 linearized vector and the GyV7 virus VP3 gene. 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.

Fig. 2 is PCR amplified GyV7 virus VP3 fragment. Lane 1, PCR product of VP3 fragment of GyV7 virus; Lane M, DNA Marker.

Figure 3 is PCR amplification of the linearized vector pGEX-6p-1. Lane 1, PCR product of linearized vector pGEX-6p-1; lane M, DNA Marker.

Fig. 4 is the expression of SDS-PAGE analysis of VP3 gene of GyV7 virus. Lane M, protein marker; lanes 1 and 2 represent the supernatant of pGEX-6P-1 sample induced by IPTG ultrasonic lysis, precipitation; lanes 3, 4, 5 represent the supernatant of VP3 sample induced by IPTG ultrasonic lysis, precipitation And purified VP3 protein.

Figure 5 is the identification of polyclonal antibodies against GyV7VP3 protein by Western blot. 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 clarify the technical scheme and technical purpose of the present invention, the present invention will be further introduced below in conjunction with the accompanying drawings and specific implementation methods.

Example 1:

1) Design and amplify the primers containing the pGEX-6p-1 linearized vector and the VP3 gene fragment of GyV7 virus:

The upstream primer for amplifying the pGEX-6p-1 linearization vector is located at positions 1022-1047 of the pGEX-6p-1 plasmid; and has an extra TAA base at the 5' end; the downstream primer for amplifying the pGEX-6p-1 linearization vector is located in pGEX -Position 916-941 of the 6p-1 plasmid; and has an extra CAT base at the 5' end. The upstream primers for amplifying the VP3 gene of GyV7 virus include the VP3 gene start codon ATG and the following 14 bases, and there are 16 reverse complementary bases at the 5' end with the downstream primer of the pGEX-6p-1 linearized vector; The downstream primer of VP3 gene of GyV7 virus includes VP3 gene termination 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 shown in Table 1, which were synthesized by Life Technologies Shanghai Thermo Fisher Scientific Co., Ltd.

Table 1: Primer design for amplification of pGEX-6p-1 linearized vector and GyV7 virus VP3 fragment:

2) PCR amplification of pGEX-6p-1 linearized vector and GyV7 virus VP3 gene fragment:

The pGEX-6p-1 plasmid and the synthesized GyV7VP3 gene were used as templates, and the primers described in Table 1 were used as primers for 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 pcGyV7-VP3 plasmid, 1 μl commercialized 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 DNAMarker, where lane 1 is the PCR product of the VP3 fragment of GyV7 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) The VP3 fragment of GyV7 virus is quickly cloned into the pGEX-6p-1 vector:

The expression linearization vector pGEX-6p-1 purified above and the PCR product of the VP3 fragment of GyV7 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 GyV7 virus VP3 fragment product, 50ng of the purified pGEX-6p-1 expression linearization vector, 2μl of commercial 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.

Example 2:

Induced expression of GyV7 virus VP3 protein and its purification: the obtained positive clone containing the GyV7 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.

The specific steps are as follows:

1) Induced expression of GyV7 virus VP3 protein:

After the obtained positive clone containing the VP3 gene of GyV7 virus (named pGEX-VP3) was transformed into BL21 bacteria, it was inoculated in 3 ml of LB liquid medium containing Amp (100 μg/mL), and cultured overnight at 37°C with shaking at 250 rpm. The next day, they were transferred to LB medium at a ratio of 1:100, shaken at 1500 rpm at 37°C for 2.5 hours, and induced with a final concentration of 1mM IPTG at 37°C. After 5 hours of induction, transfer the bacterial solution to a centrifuge tube, centrifuge at 6000rpm for 5 minutes, remove the supernatant, wash once with PBS, suspend the bacteria in 600 μL of sterilized DDW, and perform ultrasonic lysis on an ice bath, 30HZ, 10min. Centrifuge at 10,000 rpm for 5 minutes, take the supernatant, resuspend the pellet with 300 μL of sterilized DDW, and freeze it at -20°C for later use. Take 30 μL bacterial lysate supernatant and precipitate respectively, add 5 μL 6×SDS gel loading buffer and mix well, boil for 5 minutes; use SDS-PAGE (5% stacking gel, 10% separating gel) electrophoresis to analyze the expression of recombinant protein and its expression form. In Figure 4, VP3 protein can be present in soluble form in the supernatant of sonicated samples.

2) Purification of GyV7 virus VP3 protein:

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. The brief steps are as follows: add 5 volumes of DDW to the purification column to remove ethanol; equilibrate the column with 10 volumes of binding buffer (140mM Nacl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4); Repeat the column twice to fully bind the protein on the column. Use 10 volumes of binding buffer to wash the impurity protein in the column; use 5 volumes of elution buffer (50mM Tris-HCl, 10mM reduced glutathione) to elute the target protein, then add it to the dialysis bag and use 1×PBS The solution was fully dialyzed, aliquoted, and stored at -70°C; the purified protein was added in proportion to 5×Loading Buffer, boiled for 5 minutes, and analyzed by SDS-PAGE electrophoresis. The results showed that the VP3 protein was well purified (as shown in lane 5 in FIG. 4 ).

3) Immunoreactivity of GyV7 virus VP3 protein:

Purified VP3 protein was used to immunize 6-week-old BALB/C mice intraperitoneally, three times with an interval of 14 days, and the dose was 50 μg/mouse/time. For the first immunization, it was mixed with an equal volume of complete Freund's adjuvant to a water-in-oil state, and for the second immunization, it was mixed with an equal volume of incomplete Freund's adjuvant, and no adjuvant was added for subsequent immunizations. The mouse serum was collected after the third immunization, and the anti-VP3 specific antibody in the serum was analyzed by western blot using the VP3 protein expressed in 293T cells as an antigen. The results showed that the obtained mouse polyclonal antibody against GyV7 virus VP3 protein could specifically react with the VP3 protein expressed in 293T cells ( FIG. 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 GyV7 serological diagnosis.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

SEQUENCE LISTING

<110> Yangzhou University

<120> A kind of preparation method of GyV7 circular virus VP3 protein

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<170> PatentIn version 3.5

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Claims (6)

1. A method for preparing GyV7 circular virus VP3 protein, characterized in that, the method is based on the recombinase ExnaseTM II with the linearized pGEX-6p-1 carrier and the GyV7 virus VP3 gene fragment PCR product without enzyme-cut ligation reaction, direct recombination Cloning technology; and transformed into escherichia coli, realize VP3 protein expression; Wherein, the primer that is used for PCR amplification GyV7 virus VP3 gene is divided into upper and lower primers, and the nucleotide sequence of described upper primer is as shown in SEQ ID NO.3 The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. 2. a kind of GyV7 circular virus VP3 protein preparation method as claimed in claim 1, is characterized in that, specifically comprises the steps: 1) PCR amplification of the pGEX-6p-1 linearized vector and the VP3 gene fragment of the GyV7 virus: using the pGEX-6p-1 plasmid and the VP3 gene of the GyV7 virus as templates, design primers, and PCR amplify the linearized vector containing pGEX-6p-1, respectively. Gene vector and GyV7 virus VP3 gene fragment; wherein, the primers for PCR amplification of GyV7 virus VP3 gene are divided into upper and lower primers, the nucleotide sequence of the upper primer is as shown in SEQ ID NO.3, and the lower The nucleotide sequence of the primer is shown in SEQ ID NO.4; 2) Quickly clone the VP3 fragment of the GyV7 virus into the pGEX-6p-1 vector: use the recombinase ExnaseTM II for rapid recombination and cloning; 3) The positive clones were transformed into Escherichia coli to realize the expression of VP3 protein. 3. a kind of GyV7 circular virus VP3 protein preparation method as claimed in claim 2, is characterized in that, the primer that is used for PCR amplification pGEX-6p-1 linearization carrier described in step 1) is divided into upper and lower reaches Primers, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2. 4. A primer for PCR amplification of the pGEX-6p-1 linearized carrier, characterized in that, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer The sequence is shown in SEQ ID NO.2. 5. A primer for PCR amplification of the GyV7 virus VP3 gene, characterized in that the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is as in SEQ ID Shown in NO.4. 6. a kind of GyV7 circular virus VP3 albumen preparation method as described in any one in claim 1-3, and primer as claimed in claim 4 or 5 is in preparation GyV7 diagnostic antigen, anti-VP3 polyclonal antibody and preparation GyV7 Application of effective diagnostic reagents in epidemiology.