CN118726484A - ILTV gD protein antigen epitope expression cassette, recombinant virus and application - Google Patents

Abstract

The invention discloses an ILTV gD protein antigen epitope expression cassette, recombinant viruses and application thereof, and relates to the field of gene and protein engineering. The ILTV gD protein epitope expression cassette provided by the invention comprises T and B cell epitopes of gD protein from chicken infectious laryngotracheitis virus WG strain. The invention replaces the 5ab gene of the chicken infectious bronchitis virus H120 strain with the gene of the encoding ILTV gD protein epitope expression cassette to obtain the recombinant virus rH120-gD-T/B. The recombinant virus has good genetic stability, can be used for developing safe and effective IB-ILT bigeminal live vector vaccine, and solves the problem of poor stability of the current recombinant IBV.

Description

ILTV gD protein antigen epitope expression cassette, recombinant virus and application

Technical Field

The invention relates to the technical field of gene and protein engineering, in particular to an ILTV gD protein antigen epitope expression cassette, recombinant virus and application.

Background

Avian infectious bronchitis (Infectious bronchitis, IB) is an acute, highly contagious infectious disease of birds, the etiology of which is avian infectious bronchitis virus (Infectious bronchitis virus, IBV). IBVs belong to the order of the nest type (Nidovirales), the family of coronaviridae (Coronaviridae), the genus gamma-coronaviridae (Gammacoronavirus), and are circular or irregular forms of enveloped viruses. The IBV genome is a single-stranded positive strand RNA which is not segmented, about 27.6kb, and has a cap structure at the 5 'end and Poly (A) at the 3' end, and the sequence from the 5'-3' end is as follows: 5'UTR-la/1ab-S-3a-3b-E-M-5a-5b-N-3' UTR. At present, the IBV attenuated vaccine is widely used for the prevention and control of IB because of the convenient immunization mode and effectiveness. The H120 strain is the IBV vaccine strain with the widest application in the global scope in recent 60 years, the H120 strain belongs to the Mass serotype, and the safety and the effectiveness of the attenuated live vaccine obtained through continuous passage and weakening of chick embryos are widely verified.

With the intensive research on the unique transcription mechanism of coronaviruses and the development of reverse genetics systems, the development of vaccines for expressing exogenous genes by using coronaviruses as vectors is possible. Current research has found that recombinant IBV is often genetically unstable, but the stability of recombinant viruses is often maximized after the exogenous gene has replaced an unnecessary auxiliary gene. The stable expression of the foreign proteins of recombinant IBV may depend on a variety of factors including the genetic background of the vector virus and the foreign gene, the site of insertion of the foreign gene, and the importance of the viral gene to be replaced. Studies have shown that replacement of ORF3, ORF5 and gene spacer IR of the IBV genome with Renilla luciferase gene (Human Renilla Luciferase, hRluc) respectively, and the results show that the stability of recombinant IBV is highest after replacement of ORF5 with hRluc gene, but that detection of hRluc gene at nucleic acid level can only be stably inherited for 12 generations.

With the continuous development of molecular biology, the genome structure and protein function of pathogens are more deeply studied, and vaccines based on epitope begin to show good application prospects. An epitope is a specific region of an antigen molecule that is capable of specifically binding to an antibody or T cell receptor, and these regions are typically a small portion of the antigen molecule, and are critical factors in vaccine design and immunotherapy, as they induce a specific response in the immune system. Depending on the cell to which the antigen receptor binds, the antigen epitope can be classified into a T cell epitope and a B cell epitope. T cell epitopes are epitopes on antigen peptide fragments that have been processed by Antigen Presenting Cells (APCs), typically linear epitopes consisting of 8-12 amino acids, but also longer polypeptide chains. Recognition of T cell epitopes requires involvement of MHC molecules, where cd4+ T cells recognize epitopes on MHC II molecules and cd8+ T cells recognize epitopes on MHC I molecules. B cell epitopes are epitopes on intact antigen molecules that recognize the need for participation of MHC molecules, but rather exert antiviral effects by activating B lymphocytes to secrete antibodies by binding to B Cell Receptors (BCR). It has been found that shortening the length of the foreign gene generally increases the stability of the recombinant IBV. The method for constructing the recombinant IBV expressing the multiple epitopes by selecting the protective antigen epitope and taking the IBV as a carrier skeleton is an effective method for solving the problem of poor stability of the current recombinant IBV.

Infectious laryngotracheitis (Infectious laryngotracheitis, ILT) is an acute, highly contagious upper respiratory infectious disease caused by infectious laryngotracheitis virus (Infectious laryngotracheitis virus, ILTV), which was classified as a group B infectious disease by the international zooepidemicus agency (Office International des Epizooties, OIE) in 1999. Currently, the prevention and control ILT at home and abroad is mainly attenuated vaccine. Although the vaccine can prevent clinical diseases, the animal has larger response after inoculation, and the virulence return caused by latent infection is one of the important factors of ILT outbreaks, so the prevention and control of ILT have become the difficult problem of the world poultry industry.

At present, the research on the ILTV epitope is mainly focused on the gB protein, and the research and report of the epitope of the gD protein are relatively less, and the invention focuses on the research on T cell and B cell epitope of the gD protein of the ILTV WG strain, and is used for developing multi-epitope ILTV vaccine. gD glycoprotein is present on the surface of virions and is a receptor for viruses and susceptible cells to bind, and plays a key role in the processes of virus adsorption, virus-host membrane fusion and induction of protective immune responses in the body. On other herpesviruses, studies have demonstrated that BHV-1gD protein can elicit a more intense cellular immune response than gB and gC proteins, inducing more efficient neutralizing antibody production; the HSV-1gD protein induces the highest antibody level, and can completely resist the attack of the HSV virulent strain; PRV gD protein subunit vaccines are also resistant to lethal challenge of PRV. In the currently disclosed researches, chicken pox virus (Fowlpox virus, FPV) is taken as a vector to express ILTV gD glycoprotein, but the interference of maternal antibodies is a main reason that recombinant chicken pox virus live vector vaccines cannot be popularized and applied on a large scale so far, IBV is not interfered by maternal antibodies, basic immunization can be carried out at 1 day old, and the immunization blank period of chickens is shortened or eliminated, so that the recombinant chicken pox virus live vector vaccine is a good vaccine vector. In addition, the genetic engineering subunit vaccine for expressing the ILTV gD glycoprotein has better safety, but the preparation is relatively complex, additional adjuvant and enhanced injection are needed, the preparation and immunization cost is higher, the application prospect in the field of poultry vaccines is smaller, the antigenicity of the protein is influenced by the selected expression system, and a good expression system is difficult to find, so that the expression system needs to be carefully selected when the subunit vaccine is prepared. The IB-ILT bivalent live vector vaccine constructed by the invention has low preparation cost, convenient immunization mode, immunity in modes of nasal drip, eye drop, drinking water, spraying and the like, saves the use cost of clinical vaccine, has high safety, and solves the problems of large animal reaction after the current ILT attenuated vaccine inoculation and biological safety caused by latent infection.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an ILTV gD protein antigen epitope expression cassette, a recombinant virus and application, wherein the recombinant virus has good genetic stability, can provide effective protection for two epidemic diseases of IB and ILT, and solves the problem of poor stability of the current recombinant IBV.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: an ILTV gD protein epitope expression cassette is provided, which comprises at least one of 4 ILTV epitope polypeptides.

Further, the amino acid sequences of the 4 ILTV epitope polypeptides are respectively shown as SEQ ID N0.1-4.

Furthermore, flexible linker is adopted between the ILTV epitope polypeptide sequences.

Further, the amino acid sequence of the flexible linker is shown as SEQ ID N0.5.

Further, ILTV epitope polypeptides include T cell epitopes and B cell epitopes derived from gD protein of chicken infectious laryngotracheitis virus WG strain.

Further, the T cell epitope comprises an amino acid sequence with an amino acid sequence shown in at least one of SEQ ID Ns 0.6-20.

Further, the B cell epitope comprises an amino acid sequence with an amino acid sequence shown in at least one of SEQ ID Ns 0.21-25.

A gene encoding the ILTV epitope polypeptide.

A recombinant virus comprising a gene encoding the ILTV epitope polypeptide described above.

Furthermore, the recombinant virus uses the chicken infectious bronchitis virus H120 strain as a framework, and replaces the 5ab gene of the chicken infectious bronchitis virus H120 strain with the gene encoding the ILTV gD protein epitope expression cassette.

Further, the preparation method of the recombinant virus comprises the following steps: the multi-epitope chimeric gene gD-T/B of the infectious laryngotracheitis virus of the chicken is inserted into the genome of the IBV H120 strain by the Red/ET homologous recombination technology, and the obtained recombinant strain rH120-gD-T/B is saved.

A multi-epitope vaccine comprising the recombinant virus described above.

The ILTV gD protein antigen epitope expression cassette, recombinant virus or multi-epitope vaccine is applied to preparation or application of medicines for preventing infectious bronchitis and/or infectious laryngotracheitis.

In summary, the invention has the following beneficial effects:

1. The invention takes the chicken infectious bronchitis virus H120 attenuated vaccine strain as a framework, embeds the multi-epitope gene gD-T/B of the encoding chicken infectious laryngotracheitis virus into the H120 genome, and the obtained recombinant virus rH120-gD-T/B has good genetic stability, can be stably inherited to 30 generations, and solves the problem of poor stability of the current recombinant IBV.

2. The recombinant virus can be used as a bivalent vaccine candidate vaccine strain for preventing infectious bronchitis and infectious laryngotracheitis of chickens, and has important practical application value.

3. The vaccine provided by the invention is convenient to immunize in a manner of dropping nose and dropping eyes, drinking water, spraying and the like, saves the use cost of clinical vaccine, has high safety, and solves the problems of large animal response after the current ILT attenuated vaccine inoculation and biosafety caused by latent infection.

Drawings

FIG. 1 is a schematic diagram of the construction of an infectious clone of a recombinant virus of the present invention;

FIG. 2 is an electrophoretogram of intermediate vector pH 120-Delta5 ab-ccdB enzyme digestion identification;

FIG. 3 is an electrophoretogram of transcription vector pH120-gD-T/B cleavage identification;

FIG. 4 is an electrophoresis diagram of RT-PCR identification of recombinant virus rH 120-gD-T/B;

FIG. 5 shows the Western Blot identification of recombinant virus rH120-gD-T/B after infection of CK cells;

FIG. 6 is a graph showing proliferation of recombinant virus rH120-gD-T/B in chick embryos;

FIG. 7 is a graph showing the results of the genetic stability test of the exogenous gene of recombinant virus rH 120-gD-T/B.

Detailed Description

The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In the present application, it was verified that the order of T-cell and B-cell epitopes was not affected by the effect of the virus, and thus the order of T-cell and B-cell epitopes was not limited. That is, the order of T-cell and B-cell epitopes can be adjusted as desired by one skilled in the art within the scope of the present application.

EXAMPLE 1 construction of recombinant IBV chimeric to the ILTV gD protein epitope expression cassette

The transcription vector pH120-gD-T/B is prepared by embedding an ILTV gD protein antigen epitope expression cassette into the transcription vector pH120 through a two-step strategy of Red/ET recombination engineering technology and ccdB reverse screening, and the specific steps are as follows, as shown in figure 1:

Primers delta 5 ab-ccdB-F/delta 5ab-ccdB-R are designed, as shown in table 1, pBR322-ccdB-amp is taken as a template to amplify screening genes, and two ends of the amplified delta 5ab-ccdB gene fragment are provided with a section of homology arms of 5ab gene upstream and 5ab gene downstream of the H120 genome. The PCR product was detected by 1% agarose gel electrophoresis, and the target band was about 1400bp, which was consistent with the expected 1421bp size. The PCR product was recovered and purified, the concentration of the nucleic acid was measured, and a small amount was sent to a sequencing company for sequencing.

TABLE 1 design of amplification primers for screening gene ccdB

And (3) carrying out homologous recombination on the purified screening gene fragment delta 5ab-ccdB and a pH120 carrier in engineering bacteria expressing Red alpha/beta through electric conversion, culturing for 16 hours on an LB plate, and then picking single colonies for bacterial liquid PCR identification. The positive bacterial liquid is amplified and shaken to extract plasmids, and the XhoI and BstZ17I double enzyme digestion identification is carried out, and the result is shown in figure 2.

As can be seen from FIG. 2, the size of the enzyme-digested fragments of the recombinant plasmid completely accords with the preset map, and preliminary results show that the intermediate vector has the correct construction of the pH 120-delta 5 ab-ccdB.

Sequencing the digested and correctly recombinant plasmid by a sequencing company, and the result shows that all sequences are completely consistent with the expected sequence, and the construction of the intermediate plasmid pH 120-delta 5ab-ccdB is successful.

By the same method, the ILTV gD protein epitope expression cassette and an intermediate carrier pH 120-delta 5ab-ccdB are recombined, the gD-T/B epitope expression cassette replaces a screening gene ccdB, a positive bacterial liquid is identified by PCR, a plasmid is extracted after amplification and shaking, and XhoI and BstZ17I double enzyme digestion identification are carried out, and the result is shown in figure 3.

As can be seen from FIG. 3, the size of the enzyme-digested fragments of the recombinant plasmid completely accords with the preset map, and the preliminary indication shows that the recombinant plasmid pH120-gD-T/B is constructed correctly. Sequencing the recombinant plasmid with correct enzyme digestion by sequencing company, and the result shows that all sequences are completely consistent with the expected sequence, and the transcription vector pH120-gD-T/B is successfully constructed.

EXAMPLE 2 rescue and identification of recombinant Virus rH120-gD-T/B

(1) Virus rescue

The constructed transcription vector pH120-gD-T/B and helper plasmid pVAX-H120-N are co-transfected into BSR-T7/5 cells by adopting a liposome transfection method. 4h after transfection, the transfection solution was aspirated and the culture was continued with the addition of DMEM maintenance solution containing 2% FBS. Cells are harvested after 48h of transfection, cell supernatants are inoculated with 9-11 day-old SPF chick embryos after repeated freeze thawing, chick embryo allantoic fluid is harvested after 48h of incubation at 37 ℃, and recombinant virus identification is carried out after three generations of blind transmission.

(2) Recombinant virus identification

200 Mu L of virus liquid is taken to extract virus RNA according to the manual of Axyprep humoral virus DNA/RNA small extraction kit. The primer sequence and amplification length of the primer delta 5 ab-JD-F/delta 5ab-JD-R are shown in Table 2, a system is prepared according to the specification of a one-step RT-PCR Kit PRIMESCRIPT ONE STEP RT-PCR Kit Ver.2, the reaction system is shown in Table 3, and the amplification procedure is as follows: 50 ℃ for 30min;94 ℃ for 2min;94 ℃,30s,56 ℃,30s,72 ℃ and 40s for 32 cycles; 72℃for 5min. The RT-PCR products were detected by 1% agarose gel electrophoresis and sent to sequencing company for sequence sequencing, and the results are shown in FIG. 4.

TABLE 2gD-T/B multiple epitope Gene identification primer design

TABLE 3 one-step RT-PCR reaction system

Reaction components	25 Mu L of reaction system
		Primescript 1Step Enzyme Mix	0.2μL
2×1Step Buffer	12.5μL
		Upstream primer, 10. Mu.M	1μL
Downstream primer, 10. Mu.M	1μL
		RNA	2μL

As can be seen from FIG. 4, the target bands were consistent with the expected size, and the sequencing results showed 100% identity with the expected sequence, indicating successful rescue of recombinant virus rH 120-gD-T/B.

Recombinant virus rH120-gD-T/B was inoculated with a monolayer of CK cells, MOI=0.1, the medium was aspirated and removed 48h after infection, after three washes with PBS, protein lysate containing 1% protease inhibitor was added and placed on ice for 20min lysis. The whole of the lysis mixture was transferred to a 1.5mL centrifuge tube and centrifuged at 12,000Xg for 20min at 4 ℃. Sucking 80 mu L of supernatant into a new 1.5mL centrifuge tube, adding 20 mu L of 5 XSDS loading buffer, shaking and mixing uniformly, boiling for 5min, harvesting cell total proteins, and performing SDS-PAGE (Bio-Rad); transferring a protein film (Bio-Rad) onto a PVDF film, blocking with 5% skim milk overnight, washing with PBST (0.05% Tween 20), adding a primary antibody solution diluted according to a specific ratio, and incubating for 1h at room temperature; the primary antibody solution was discarded and the membrane was washed 3 times with 5min each with PBST on a room temperature shaker. Adding a secondary antibody solution diluted according to a specific proportion, and incubating for 1h at room temperature; the secondary antibody solution was discarded and the membrane was washed 3 times with 5min each with PBST on a room temperature shaking table. And (3) placing the PVDF film on an imaging system operation table, dropwise adding the hypersensitive ECL chemiluminescent liquid on the film, and adjusting instrument parameters to expose. The results are shown in FIG. 5.

As can be seen from FIG. 5, recombinant virus rH120-gD-T/B successfully expressed gD-T/B multi-epitope protein.

EXAMPLE 3 proliferation Properties and stability of recombinant Virus rH120-gD-T/B

(1) Proliferation curve determination

To determine the proliferation curve of recombinant virus on chick embryos, rH120-gD-T/B and the parental virus H120 strain were diluted with physiological saline and inoculated with 10 day old SPF chick embryos, 0.1 mL/embryo, by the allantoic route at an inoculum size of 100EID ₅₀. Chick embryo allantoic fluid was harvested 12h, 24h, 36h, 48h, 60h and 72h after inoculation, respectively, and EID ₅₀ measurements were performed. The results are shown in FIG. 6.

As can be seen from FIG. 6, the growth kinetics of recombinant virus rH120-gD-T/B chick embryo is similar to that of the maternal virus H120 strain, and the proliferation characteristics of high titer growth on chick embryo are still maintained.

(2) Stability detection

Recombinant virus rH120-gD-T/B was continuously transferred to the chick embryo for 30 generations, and RT-PCR was performed with the virus solutions of the P5, P10, P15, P20, P25 and P30 generations, and the primers were referred to Table 2. The results are shown in FIG. 7.

As can be seen from FIG. 7, all the generation of subgroup viruses rH120-gD-T/B can amplify bands consistent with the expected size, which indicates that the recombinant viruses can stably pass to at least the 30 th generation on the chick embryo.

EXAMPLE 4 toxicity protection experiment of recombinant Virus rH120-gD-T/B

40 SPF chickens of 1 day old were randomly divided into 4 groups of 10 animals each, and recombinant virus rH120-gD-T/B and PBS were inoculated by the nasal drop eye drop route at an immunization dose of 10 ^4.0EID₅₀/animal. The IBV M41 strain and ILTV WG strain were challenged at 14 and 28 days of age, respectively, and the specific groupings and experimental procedures are shown in table 4.

Table 4 experimental grouping and procedure

As can be seen from Table 4, the experiment of virulent M41 strain virus attack of chicken infectious bronchitis virus was performed 14 days after immunization, and continuous observation was performed for 10 days, wherein the incidence of the virus attack control group was 100%, the incidence of the rH120-gD-T/B immune group was 10%, and the sick chicken showed cough, head throwing, mental depression, feather disorder, typical sound-throwing, mouth opening respiration and the like. And (3) carrying out a virulent WG strain virus challenge experiment of the infectious laryngotracheitis virus of the chicken 28 days after immunization, continuously observing for 10 days, wherein the incidence of a virus challenge control group is 100%, the incidence of an rH120-gD-T/B immune group is 0, and the specific symptoms of the sick chicken are cough, head throwing, mouth opening breathing, hemoptysis, death and the like.

In conclusion, after the recombinant vaccine rH120-gD-T/B provided by the invention is used for immunizing SPF chickens, the attack of IBV virulent and ILTV virulent can be effectively resisted, and 90% and 100% protection rates are respectively provided, so that the bivalent carrier vaccine is proved to be safe and effective.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims (9)

1. The ILTV gD protein epitope expression cassette is characterized by comprising at least one of 4 ILTV epitope polypeptides, wherein the amino acid sequences of the 4 ILTV epitope polypeptides are respectively shown as SEQ ID N0.1-4.
2. 2. The ILTV gD protein antigen epitope expression cassette of claim 1, wherein the ILTV antigen epitope polypeptide sequences are connected by a flexible linker, and the amino acid sequence of the flexible linker is shown as SEQ ID N0.5.
3. 3. The ILTV gD protein epitope expression cassette of claim 1 or 2, wherein the ILTV epitope polypeptide comprises a T cell epitope and a B cell epitope of gD protein derived from chicken infectious laryngotracheitis virus WG strain.
4. 4. The ILTV gD protein antigen epitope expression cassette of claim 3, wherein the T cell epitope comprises an amino acid sequence as set forth in at least one of SEQ ID nos 0.6-20 and the B cell epitope comprises an amino acid sequence as set forth in at least one of SEQ ID nos 0.21-25.
5. 5. A gene encoding an ILTV epitope polypeptide as claimed in any one of claims 1to 3.
6. 6. A recombinant virus comprising the gene of claim 5.
7. 7. The recombinant virus according to claim 6, wherein the recombinant virus uses chicken infectious bronchitis virus H120 strain as a skeleton, and replaces the 5ab gene of chicken infectious bronchitis virus H120 strain with a gene encoding an ILTV gD protein epitope expression cassette.
8. 8. A multi-epitope vaccine comprising the recombinant virus of claim 6.
9. 9. Use of an ILTV gD protein antigen epitope expression cassette according to any one of claims 1-4, a recombinant virus according to any one of claims 6-7 or a multi-epitope vaccine according to claim 8 for the preparation or use as a medicament for the prevention of infectious bronchitis and/or infectious laryngotracheitis in chickens.