CN110747215A - Recombinant baculovirus for efficiently expressing hog cholera E2 protein and construction method thereof - Google Patents

Abstract

The invention relates to a recombinant baculovirus for efficiently expressing hog cholera E2 protein and a construction method thereof, the method firstly synthesizes a sequence which comprises a translation enhancer sequence TE21 with 21 bases, a human albumin signal peptide sequence S with 54 bases and an E2 sequence without a transmembrane region, and simultaneously, a 6his sequence is added behind the E2 sequence for convenient detection and purification. The sequence is connected to the downstream of a p10 promoter of pFastbac-dual to construct a swine fever E2 recombinant baculovirus expression vector, a bacmid is prepared by transforming DH10Bac, and the bacmid is transfected into an insect cell SF9 to obtain a recombinant baculovirus E2-rBV capable of efficiently secreting E2 protein. The recombinant baculovirus E2-rBV has raised E2 protein expression amount, and may be secreted into culture supernatant and cell crushing liquid supernatant simultaneously to facilitate the glycosylation, correct folding and purification of E2 protein.

Description

Recombinant baculovirus for efficiently expressing hog cholera E2 protein and construction method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant baculovirus capable of efficiently expressing hog cholera E2 protein and a construction method thereof.

Background

Classical Swine Fever Virus (CSFV) brings great economic loss to the breeding industry because of the super-strong infectivity, rapid propagation speed and wide distribution range. The plan for eliminating the swine fever is put forward in 1956 in China, and the swine fever is still continuously popular in China nowadays. The swine fever vaccines sold in the market at present are all traditional vaccines including swine fever inactivated vaccines and swine fever attenuated live vaccines. Although the whole virus vaccine has good immune effect, the whole virus vaccine has the risks of incomplete inactivation, high toxicity, occurrence of new strains caused by gene mutation and the like, and in addition, the whole virus vaccine cannot distinguish immune infection from wild virus infection, so that the purification of the swine fever is difficult. With the increasing urgency of swine fever decontamination, the subunit vaccine of swine fever virus completely avoids the disadvantages and becomes a focus of future markets.

CSFV is an enveloped single-stranded positive-stranded RNA virus containing only one large Open Reading Frame (ORF), in which the structural protein E2 has a molecular weight of 55kDa and is the least conserved, most variable molecule. E2 is also the most major immunoprotective protein. In the process of virus infection entering cells, E2 protein with a certain concentration can inhibit the mediation effect of virus entering cells and block the infection of CSFV to in vitro cultured cells. The CSFV E2 protein expressed and purified by the virus as a vector or other genes can protect the immune pig against the infection of CSFV. Recent studies have shown that glycoprotein E2 is a determinant of virulence.

At present, a classical swine fever virus E2 protein recombinant baculovirus inactivated vaccine, Tiankang biological corporation, has obtained a new veterinary certificate. CN 106139139A discloses preparation of a subunit vaccine of swine fever E2 genetic engineering, wherein a utilized baculovirus expression vector is pFastBac 1 for expression and purification of E2 protein, and a GP67 signal peptide sequence is added in front of an E2 sequence. CN 106110319A discloses a preparation method of a swine fever virus E2 gene recombinant baculovirus inactivated vaccine. The soluble E2 protein was obtained by the addition of the MELs signal peptide, and the expression vector was pFastBac-dual. CN 109182380A discloses a preparation method and application of a baculovirus expressed classical swine fever E2 subunit vaccine, wherein a classical swine fever E2 protein containing a signal peptide is utilized, and an expression vector is pFastBacTM-HTB. In the development process of the subunit vaccine of the swine fever E2 gene engineering, the expression level and solubility of the E2 protein are one of the factors which restrict the industrialization of the swine fever E2 gene engineering.

Disclosure of Invention

In order to solve the problems in the prior art, the invention designs a construction method of a recombinant baculovirus for efficiently expressing hog cholera E2 protein. Firstly, the invention artificially synthesizes a gene sequence for constructing a recombinant baculovirus for efficiently expressing the hog cholera E2 protein, wherein the sequence comprises a translation enhancer sequence TE21 rich in AT basic groups, and the size of the translation enhancer sequence TE21 is 21 basic groups; a human albumin signal peptide sequence S, which codes 17 amino acids MKWVTFISLLFLFSSAY and has the size of 51 basic groups; and a hog cholera E2 sequence with a size of 1011 bases, wherein the transmembrane region is removed; and 6 histidine his sequences, 18 bases in size, with XhoI and KpnI cleavage sites at each end of the sequence. The gene sequence is shown in SEQ ID NO.1 and is named as: TE21-S-E2-6 His.

Further, the enhancer sequence TE21 is shown as SEQ ID NO.2, and the signal peptide sequence S is shown as SEQ ID NO. 4.

Meanwhile, the invention designs a vector for expressing the gene sequence, the expression vector recovers a connecting fragment and a vector fragment through an XhoI and KpnI enzyme digestion sequence TE21-S-E2-6His and a vector pFastbac-dual, converts DH5 α, and constructs the vector through clone PCR verification, sequencing verification and verification, wherein the vector is named as TE21-S-E2-6 His-pFastbac-dual.

The invention also discloses a recombinant baculovirus for efficiently expressing the hog cholera E2 protein, which is transfected by a plasmid carrying the gene sequence and is named as TE21-S-E2-rBV (rBV-DC-1 strain), and is preserved in the China general microbiological culture Collection center at 19 months 9 and 2019 with the preservation number of CGMCC No. 18508.

The construction method of the recombinant baculovirus for efficiently expressing the hog cholera E2 protein comprises the following steps of (1) artificially synthesizing the TE21-S-E2-6His sequence of claim 1;

(2) XhoI and KpnI enzyme digestion TE21-S-E2-6his sequence and pFastbac-dual vector sequence, recovering fragments syn-S-E2-6his and pFastbac-dual, connecting, transforming, cloning, sequencing and verifying, and constructing a TE21-S-E2-6his-pFastbac-dual expression vector;

(3) transforming a TE21-S-E2-6his-pFastbac-dual expression vector into DH10Bac, and obtaining recombinant bacmid TE21-S-E2-6his-rBacmid through blue-white spot screening and PCR identification;

(4) transfecting recombinant bacmid TE21-S-E2-6his-rBacmid to SF9 cells, harvesting P1-generation baculoviruses, subculturing to P3 generation, and carrying out expression verification on E2 protein on the P3-generation baculoviruses to obtain the recombinant baculovirus capable of efficiently expressing the swine fever E2 protein, wherein the recombinant baculovirus is named as TE21-S-E2-rBV strain.

Compared with the prior art, the recombinant baculovirus for efficiently expressing the swine fever E2 protein disclosed by the invention has the advantages that a gene sequence of the swine fever E2 protein, which contains a translation enhancer sequence TE21, a human albumin signal peptide sequence S and no transmembrane region, is provided, and the sequence is inserted into a baculovirus expression vector to provide powerful support for efficient expression of the swine fever E2 protein in the baculovirus; the expression level of the E2 protein of the recombinant baculovirus obtained by the construction method designed by the invention is obviously improved, the secretion of the protein is facilitated, the E2 protein can be detected in culture medium supernatant and cell disruption liquid supernatant, the glycosylation and the correct folding and purification of the E2 protein are facilitated, and the immunogenicity of the E2 protein in a pig body can be improved through the glycosylation and the correct folding of the E2 protein, so that the E2 protein can be well applied to the research and development of a classical swine fever virus subunit vaccine.

Drawings

FIG. 1 shows the WB detection results of example 4

FIG. 2 is the level of antibody after Elisa test of E2 subunit vaccine immunization of mice in example 5

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific embodiments, which are only used for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.

The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

The list of agents, strains and plasmid sources of the invention is as follows:

SF9 cells were purchased from Invitrogen corporation, USA

pFastbac-dual was purchased from Invitrogen corporation, USA

Cell culture media was purchased from Gibco, USA

Transfection reagent Expifeacylamine SF was purchased from Gibco, USA

Sequences and primers were synthesized in Biotechnology engineering (Shanghai) Ltd

PCR polymerase and restriction enzyme were purchased from NEB of UK

DH10Bac competence purchased from Beijing Soilebao

ISA201 adjuvant purchased from Saibox, France

Example 1

Synthesis of TE21-S-E2-6His sequence

The TE21-S-E2-6His sequence is synthesized by referring to the E2 gene sequence of the domestic classical strain C strain, the sequence comprises a translation enhancer sequence TE21 rich in AT base, the size of the translation enhancer sequence TE21 is 21 bases, and the base sequence is shown as SEQ ID NO. 2; a human albumin signal peptide sequence S with the size of 51 basic groups and the nucleotide sequence shown in SEQ ID NO.4, wherein the signal peptide sequence S codes 17 amino acids and the amino acid sequence shown in SEQ ID NO. 5; and a truncated E2 sequence of hog cholera E2 protein, the size of which is 1011 bases; and 6 histidine his sequence with 18 bases in size, and XhoI and KpnI cutting sites in the two ends of the sequence. The synthesized TE21-S-E2-6His sequence is shown in SEQ ID NO. 1. The protein sequence coded by the S-E2-6His sequence is shown in SEQ ID NO. 3.

Example 2

Baculovirus expression vector construction

The TE21-S-E2-6His sequence synthesized in example 1 and a purchased pFastbac-dual vector sequence are digested by XhoI and KpnI, a syn-S-E2-6His and pFastbac-dual sequence fragment after digestion are recovered, and are subjected to ligation, transformation, cloning, sequencing verification to construct a TE21-S-E2-6His-pFastbac-dual expression vector.

Meanwhile, a primer is designed, TE21-S-E2-6His-pFastbac-dual is used as a template, a sequence S-E2-6His without an enhancer and a sequence E2-6His without the enhancer and a signal peptide are amplified, and XhoI and KpnI enzyme cutting sites are added at both ends of the sequence. S-E2-6His-pFastbac-dual expression vector without translation enhancer and E2-6His-pFastbac-dual expression vector without signal peptide and enhancer are constructed by using XhoI and KpnI enzyme digestion S-E2-6His and E2-6His sequence and pFastbac-dual vector through connection, transformation and screening cloning methods. PCR, enzyme digestion, connection, transformation and the like are all conventional methods in molecular biology.

Example 3

Preparation of bacon

Three expression vectors TE21-S-E2-6 his-pFastdeal, S-E2-6 his-pFastdeal and E2-6 his-pFastdeal prepared in example 2 are respectively transformed into competent cells DH10Bac, after the cells are cultured at 37 ℃, three groups of cells are screened by using a blue white spot screening method and are subjected to PCR identification, and three recombinant bacmid TE21-S-E2-6his-rBacmid, S-E2-6his-rBacmid and E2-6his-rBacmid are obtained. Bacmid transformation and screening methods refer to Invitrogen company Bac-to-Bac^TMThe baculovirus expression system operates in the user guide.

Example 4

Recombinant baculovirus harvest and E2 protein expression identification

Three recombinant bacmids of TE21-S-E2-6his-rBacmid, S-E2-6his-rBacmid and E2-6his-rBacmid obtained in example 3 were transfected with SF9 cells in logarithmic growth phase respectively by using transfection reagent Expifeacylamine SF, and after 72h of culture, recombinant baculovirus TE21-S-E2-6his-rBV, S-E2-6his-rBV and E2-6his-rBV of P1 generation were harvested respectively. Wherein the TE21-S-E2-rBV (rBV-DC-1 strain) is classified as polyhedrosis virus (Nucleopolyhedovirus), is preserved in the China general microbiological culture Collection center on 19 months and 9 months in 2019, is preserved at the microbial research institute of China academy of sciences No.3 of West Lu 1 of the morning of the Yangyang district in Beijing city, and has the preservation number of CGMCC No. 18508.

The harvested three groups of P1 generation recombinant baculoviruses were continuously subcultured on sf9 cells to P3 generation, the P3 generation viruses were centrifuged, the supernatant was virus solution, and the P3 generation virus titer was determined by plaque method. Infecting SF9 cells with P3 substitute virus according to the inoculation amount of 0.1MOI, culturing for 96h, centrifuging, collecting supernatant as virus solution, repeatedly freezing and thawing the precipitate and performing ultrasonic treatment, centrifuging, collecting cell supernatant, and dissolving the cell precipitate with PBS. And simultaneously carrying out SDS-PAGE and WB detection on the virus liquid, the cell supernatant and the cell precipitation liquid. The detection results are shown in fig. 1 and table 1:

TABLE 1 molecular weight and protein yield tabulation of baculovirus-expressed E2 proteins constructed from different vectors

In Table 1 and FIG. 1, 1 is a positive control, and 2, 3 and 4 are cell pellet, cell supernatant and virus supernatant of E2-6his, respectively; 5. 6 and 7 are cell sediment, virus liquid supernatant and cell supernatant of TE21-S-E2-6his respectively; 11. 12 and 13 are cell sediment, cell supernatant and virus liquid supernatant of S-E2-6 his. In addition, in FIG. 1, 8, 9 and 10 are the supernatant of the control of the empty cells, the supernatant and the pellet after the empty cells are disrupted.

As can be seen from FIG. 1 and the above table, TE21-S-E2-6his-rBV, S-E2-6his-rBV and E2-6his-rBV three groups of recombinant baculovirus are compared, as TE21-S-E2-6his-rBV contains a translation enhancer sequence, the expression of E2 protein is enhanced, and the expression quantity of E2 is higher than that of S-E2-6his-rBV and signal peptide E2-6 his-rBV; meanwhile, TE21-S-E2-6his-rBV and S-E2-6his-rBV contain signal peptide S sequences, and the amount of E2 in the supernatant of virus liquid and the supernatant of cells is higher than that in cell precipitates, which shows that the S sequences promote the secretion of proteins, the proteins can be fully glycosylated, the molecular weight of the proteins is about 55Kd, and the protein expression amount of the group E2-6his-rBV without adding signal peptide and translation enhancer is low, and the molecular weight is about 48 Kd. Therefore, the signal peptide can promote the E2 protein to be secreted into cell supernatant and culture medium supernatant, and simultaneously can guide the protein to endoplasmic reticulum and Golgi apparatus, and further carry out glycosylation, phosphorylation and other modifications on the protein, so that the protein is closer to the structure of the protein in a virus body; and the translation enhancer TE21 can enhance the expression of the E2 protein.

The assay purified E2 protein by nickel column affinity chromatography using his tag and protein concentration was determined by BCA method. As can be seen from the data in Table 1, the E2 protein content in the supernatant of TE21-S-E2-6his-rBV cell sap is about 75. mu.g/ml, the E2 protein content in the supernatant of S-E2-6his cell sap is about 30. mu.g/ml, the E2 protein content in the supernatant of E2-6his-rBV cell sap is only 5. mu.g/ml, and the E2 expression level in TE21-S-E2-6his-rBV is 2.5 times that of E2 expression level in S-E2-6his-rBV and 13 times that of E2-6 his-rBV. The total protein content TE21-S-E2-6his-rBV of E2 is 2.38 times of the expression amount of E2 in S-E2-6his-rBV and 7.75 times of E2-6 his-rBV.

Example 5

Preparation of E2 subunit vaccine and mouse immunity test

The three strains of TE21-S-E2-6his-Rbv, S-E2-6his-rBV and E2-6his-rBV obtained in example 4 were amplified to the 4 th generation, virus titer was measured, SF9 insect cells were transfected at an inoculation amount of 0.1MOI, suspension culture was continued, and the cells were cultured at 27 ℃ and 150rpm for 96 hours to harvest. The yield of E2 protein was calculated by SDS-PAGE and BCA. Simultaneously, the inactivating agent BEI is added into the antigen with the final concentration of 1mmol/L and is inactivated at 37 ℃ for 48 hours. 1:1 and an adjuvant ISA201 to prepare the classical swine fever virus E2 recombinant baculovirus inactivated vaccine with the unified antigen content of 50 mug/mL.

40 BALB/c 5-6 week female mice were divided into 4 groups of 10 mice each, 1 group was TE21-s-E2 subunit recombinant baculovirus inactivated vaccine, 2 groups was s-E2 subunit recombinant baculovirus inactivated vaccine, 3 groups was E2 subunit recombinant baculovirus inactivated vaccine, and 4 groups was placebo group. The vaccine is injected into the inner thigh muscle of the mouse at the injection time of 0d and 14d, the injection amount is 10 mu g/head, and the blood sampling time is 0d, 14d and 28 d. The antibody content was detected by indirect ELISA. The results of the tests are shown in FIG. 2 and Table 2,

TABLE 2 ELISA determination of mouse serum antibody content

The results show that the serum antibodies of the mice in the TE21-s-E2 and s-E2 groups are equivalent, but are higher than those of the mice in the E2 group and are obviously higher than those of the placebo group. The reason for the analysis is that the protein molecular weight of the E2 antigen of the TE21-s-E2 and s-E2 groups is 55kD, the protein glycosylation is sufficient, the protein structure is closer to the original virus structure, and therefore, the antigen has better immunogenicity and can induce higher antibody production. And the molecular weight of the E2 antigen of the E2 group is 48kD, and glycosylation is insufficient, so that the immunogenicity is poor, and the antibody titer is low.

The above description is only for the preferred embodiment of the present invention, and should not be taken as limiting the scope of the invention, which is defined by the appended claims and the description of the invention.

Sequence listing

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Claims (5)

1. An artificially synthesized gene sequence, which is characterized by comprising a translation enhancer sequence TE21 rich in AT basic groups, a human albumin signal peptide sequence S, an E2 sequence without a transmembrane region and a 6His sequence of 6 histidines, wherein the sequence is shown as SEQ ID NO.1 and is named as TE21-S-E2-6 His.

2. The gene sequence of claim 1, wherein the enhancer sequence TE21 is shown in SEQ ID NO.2, and the signal peptide sequence S is shown in SEQ ID NO. 4.

3. An expression vector is characterized in that the expression vector is constructed by recycling a connecting fragment through XhoI and KpnI enzyme digestion sequences TE21-S-E2-6His and a vector pFastbac-dual, screening, verifying and cloning, and is named as TE21-S-E2-6 His-pFastbac-dual.

4. A recombinant baculovirus for efficiently expressing classical swine fever E2 protein is characterized in that the recombinant baculovirus is transfected by a plasmid carrying the gene sequence of claim 1, is named as TE21-S-E2-rBV strain, is preserved in the China general microbiological culture Collection center (CGMCC) in 2019, 9 and 19 days, and has the preservation number of CGMCC No. 18508.

5. The method for constructing the recombinant baculovirus of claim 4 for efficiently expressing classical swine fever E2 protein, which comprises the steps of (1) artificially synthesizing the TE21-S-E2-6His sequence of claim 1;

(2) XhoI and KpnI enzyme digestion TE21-S-E2-6his sequence and pFastbac-dual vector sequence, recovering fragment syn-signal-E2-6his and pFastbac-dual, connecting, transforming, cloning, sequencing and verifying, and constructing TE21-S-E2-6his-pFastdual expression vector;

(3) transforming a TE21-S-E2-6his-pFastbac-dual expression vector into DH10Bac, and obtaining recombinant bacmid TE21-S-E2-6his-rBacmid through blue-white spot screening and PCR identification;

(4) transfecting the recombinant bacmid TE21-S-E2-6his-rBacmid into SF9 cells, collecting the virus, subculturing the recombinant bacmid to obtain the recombinant baculovirus capable of efficiently expressing the swine fever E2 protein, and naming the recombinant baculovirus as a TE21-S-E2-rBV strain.

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