Vaccine for highly expressing porcine alpha interferon and PRRSV GP5 protein as well as preparation method and application of vaccine

Abstract

The invention provides a vaccine for high expression of porcine interferon alpha and PRRSV GP5 protein, a preparation method and application thereof, and belongs to the technical field of medical preparations, the nucleic acid artificial sequence of the PRRSV GP5 protein is as shown in SEQ ID NO.1, and the nucleic acid artificial sequence of the porcine interferon alpha is as shown in SEQ ID NO.3. The invention further provides a preparation method of the vaccine for high expression of the porcine interferon alpha and the PRRSV GP5 protein. According to the invention, PRRSV GP5 protein, self-splitting polypeptide T2A and porcine alpha interferon genes are connected in series for the first time and then are stably integrated into a DC cell genome to prepare the DC vaccine, the vaccine can continuously express a PRRSV GP5 antigen, and the antigen can continuously stimulate and activate DC and activate cellular immunity and humoral immunity at the same time to play an anti-PRRSV role; the vaccine can release porcine alpha interferon and directly inhibit PRRSV infection.

Description

Technical Field

[0001] The invention relates to a vaccine for highly expressing porcine interferon-alpha and PRRSV GP5 protein, a preparation method and application thereof, and belongs to the technical field of medical preparations. Background Art

[0002] Dendritic cells (DC) are the most powerful antigen-presenting cells discovered so far. They have abundant antigen-presenting molecules on their surface and can effectively activate naive T cells. DC can also regulate memory B cells and plasma cells through IL-6 and IFN-γ, thereby affecting humoral immunity. DC vaccine is a new type of biological immunotherapy. It is different from traditional inactivated or attenuated vaccines. It is a vaccine prepared by DC-loaded antigen peptides. It currently plays an important role in the fields of human anti-tumor and anti-virus. However, it is in a blank stage in animal husbandry research.

[0003] Interferon (IFN) is a multifunctional secretory protein produced by the animal body, which has biological functions such as antiviral, anti-tumor, and immune regulation. Interferon alpha (IFN-α) is mainly produced by mononuclear phagocytes, and is a bridge between natural immunity and acquired immunity. It can activate the cytotoxicity of NK cells and promote their proliferation, regulating the body's immunity. It has antiviral effects and can be used in combination with vaccines to enhance the immune effect, or it can be used alone to improve the immune function of animals and treat viral diseases. However, the antiviral activity of recombinantly expressed monomeric interferon is not high, and it cannot have both antiviral and immune regulatory effects.

[0004] Porcine Reproductive and Respiratory Syndrome (PRRS) is an acute viral infectious disease caused by the infection of pigs with the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). PRRSV can infect pigs of all ages, especially pregnant sows and piglets, where clinical symptoms are the most severe. Due to the genetic diversity of PRRSV strains, the currently available inactivated vaccines and live attenuated vaccines can only provide preventive effects but not therapeutic effects, and live attenuated vaccines have the risk of reversion to virulence and genetic instability. Therefore, it is urgent to develop other effective PRRS prevention and control methods.

[0005] There is no report in the prior art on combining porcine interferon-α with PRRSV GP5 protein to prepare a vaccine against PRRS. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a vaccine that highly expresses porcine interferon-α and PRRSV GP5 protein, a preparation method and its application, to achieve the following invention objectives: to improve the antiviral effect of the vaccine on porcine reproductive and respiratory syndrome virus, and to improve the therapeutic effect of the vaccine on porcine reproductive and respiratory syndrome.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: A vaccine highly expressing porcine interferon-alpha and PRRSV GP5 protein, wherein the artificial nucleic acid sequence of the PRRSV GP5 protein is shown as SEQ ID NO.1, and the artificial nucleic acid sequence of the porcine interferon-alpha is shown as SEQ ID NO.3.

[0008] The vaccine contains a target gene fragment, which is obtained by connecting PRRSV GP5 protein, self-cleaving polypeptide T2A and porcine alpha interferon in series.

[0009] The artificial nucleic acid sequence of the self-cleaving polypeptide T2A is shown in SEQ ID NO.2.

[0010] The vaccine is a mature DC cell containing a target gene fragment.

[0011] The vaccine is mature DC cells infected with a recombinant lentivirus containing a target gene fragment.

[0012] The preparation method of the vaccine comprises the following steps: inserting a target gene fragment into a lentiviral expression vector to construct a target expression plasmid, packaging the lentivirus, infecting immature DC cells, and then inducing culture to obtain mature DC cells.

[0013] The vaccine is used in preparing medicines for preventing or treating porcine reproductive and respiratory syndrome.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention is the first to connect the genes of PRRSV GP5 protein, self-cleaving polypeptide T2A and porcine alpha interferon in series, and then stably integrate them into the DC cell genome to prepare a DC vaccine. The vaccine can continuously express PRRSV GP5 antigen, which can continuously stimulate and activate DC, while activating cellular immunity and humoral immunity to exert an anti-PRRSV effect; the vaccine can release porcine alpha interferon to directly inhibit the infection of PRRSV.

[0015] The present invention optimizes the nucleotide sequences of PRRSV GP5 protein and porcine interferon alpha, which can improve the immune effect of DC vaccine, improve the antiviral effect on PRRSV, and improve the therapeutic effect on porcine reproductive and respiratory syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 An inverted microscope image and a partial inverted microscope magnified image of mDC cells infected with the optimized recombinant lentivirus prepared in the present invention; A is an inverted microscope image of mDC cells infected with the optimized recombinant lentivirus, and B is a local inverted microscope magnified image; Figure 2 The figure is a flow cytometric graph showing the expression of maturation markers in DC cells, imDC cells, and mDC cells infected with optimized recombinant lentivirus. A is a flow cytometric graph of the expression of CD11C, a maturation marker of the three cells; B is a flow cytometric graph of the expression of CD86, a maturation marker of the three cells; C is a flow cytometric graph of the expression of CD80, a maturation marker of the three cells; Figure 2 The red color in the middle represents the expression of maturation markers of DC cells that were transfected with empty vectors, the blue color represents the expression of maturation markers of imDC cells, and the yellow color represents the expression of maturation markers of mDC cells that were infected with optimized recombinant lentivirus; Figure 3 Western Blotting diagram of the expression of PRRSV GP5 and Po-IFN-α in DC cells transfected with empty vector, mDC cells infected with recombinant lentivirus before optimization, and mDC cells infected with recombinant lentivirus after optimization; In the figure, 1 is the expression bands of PRRSV GP5 and Po-IFN-α of DC cells transfected with empty vector, 2 is the expression bands of PRRSV GP5 and Po-IFN-α of mDC cells infected with recombinant lentivirus before optimization, and 3 is the expression bands of PRRSV GP5 and Po-IFN-α of mDC cells infected with recombinant lentivirus after optimization. DETAILED DESCRIPTION

[0017] Example 1 Preparation of vaccine 1. Insert the gene fragment PRRSV GP5-T2A-Po-IFN-α into the lentiviral expression vector pLenti-C-GFP

[0018] The optimized artificial nucleic acid sequence of PRRSV GP5 (as shown in SEQ ID NO.1), the artificial nucleic acid sequence of self-cleaving polypeptide T2A (as shown in SEQ ID NO.2), and the optimized artificial nucleic acid sequence of Po-IFN-α (as shown in SEQ ID NO.3) were sequentially connected in series, and BamHI and NotI restriction sites were added at both ends to obtain the optimized artificial nucleic acid sequence of PRRSV Gp5-T2A-Po-IFN-α.

[0019] GenScript Biotech was commissioned to synthesize the above-mentioned optimized artificial nucleic acid sequence of PRRSV GP5-T2A-Po-IFN-α, which was inserted into the lentivirus pLenti-C-GFP vector after enzyme digestion and transformed into E. coli (DH5α). After nucleic acid sequencing confirmed that it was correct, the plasmid was extracted and purified using Qiagen's plasmid purification kit to obtain the optimized target expression plasmid.

[0020] 2. Recombinant Lentivirus Packaging The lentivirus packaging cell line 293T was inoculated into a culture dish containing DMEM medium containing 10 vol% FBS, cultured at 37°C and 5% CO2, and transfected after the adhesion rate reached 80%.

[0021] The lentiviral packaging plasmid and the optimized target expression plasmid were co-transfected into 293T cells by calcium phosphate transfection. 24 hours after transfection, the cells were significantly enlarged and spherical, the nucleus became larger and rounder, the adhesion ability decreased and it was easy to fall off; 48 hours after transfection, green fluorescent protein expression was observed in the cells under an inverted fluorescence microscope; 72 hours after transfection, the supernatant was collected and filtered to obtain the optimized recombinant lentivirus, which was stored in a -80℃ low-temperature refrigerator for future use.

[0022] The virus titer was measured by the lentivirus titer rapid test card (Antaiji (Beijing) Biotechnology Co., Ltd.), and the results showed that the titer of the optimized recombinant lentivirus was 2.79×10 6 pfu / mL.

[0023] 3. Induction of immature DCs (imDCs) 100 mL of piglet umbilical cord blood was collected and mononuclear cells were separated using lymphocyte separation fluid. 6 The mononuclear cells were inoculated at an amount of 1 cell / mL in a culture flask containing a culture medium (purchased from TaKaRa, GT-T551). After culturing for 3 hours at 37°C and 5% CO2, the suspended T cells were discarded, the adherent cells were retained, and a culture medium containing cytokines pIL-4 (final concentration of 50 ng / mL) and pGM-GSF (final concentration of 100 ng / mL) (purchased from TaKaRa, GT-T551) was added to induce the mononuclear cells to differentiate into imDCs. New culture medium was replaced every 48 hours, and imDCs were obtained on the 5th day of culture.

[0024] 4. Infection of imDCs with Recombinant Lentivirus Set imDC to 1×10 6 The cells / well were inoculated in a 12-well culture plate, and the MOI was controlled to be 15. The imDCs were infected with the optimized recombinant lentivirus obtained in step 2. After 12 hours of infection, the cells were washed 3 times with PBS to obtain imDCs infected with the optimized recombinant lentivirus.

[0025] 5. Induce mature DC cells ImDCs infected with the optimized recombinant lentivirus were cultured at a rate of 1×10 6 The cells were inoculated in a culture medium containing the maturation factor pTNF-α (final concentration of 100 μg / mL) (purchased from TaKaRa, GT-T551) and cultured for 36 h to induce mDC cells (mature DC cells) infected with the optimized recombinant lentivirus.

[0026] The morphology of mDC cells infected with the optimized recombinant lentivirus was observed under an inverted microscope. Figure 1 ,Depend on Figure 1 It can be seen that the surface of mDC cells has dendrite-like protrusions with irregular shapes, and the cells are large and translucent.

[0027] The expression of maturation markers in DC cells, imDC cells, and mDC cells infected with optimized recombinant lentivirus was analyzed by flow cytometry. Figure 2 It can be seen that the mDC cells infected with the optimized recombinant lentivirus can highly express CD11C, CD80 and CD86.

[0028] Western Blotting was used to detect the expression of PRRSV GP5 and Po-IFN-α in mDC cells infected with the optimized recombinant lentivirus. The results showed that the mDC cells infected with the optimized recombinant lentivirus could highly express PRRSV GP5 and Po-IFN-α proteins (see Figure 3 ).

[0029] The DC cells were transfected with empty vectors by using the lentivirus pLenti-C-GFP vector to transfect 293T cells according to the method of Example 1 to obtain lentivirus, which was then infected with imDC to induce mature DC cells.

[0030] The artificial nucleic acid sequence of PRRSV GP5 before optimization (as shown in SEQ ID NO.4), the artificial nucleic acid sequence of self-cleaving polypeptide T2A (as shown in SEQ ID NO.2), and the artificial nucleic acid sequence of Po-IFN-α before optimization (as shown in SEQ ID NO.5) were sequentially connected in series, and NotI and XhoI restriction sites were added at both ends to obtain the artificial nucleic acid sequence of PRRSV GP5-T2A-Po-IFN-α before optimization. The target expression plasmid before optimization was prepared according to the above method of Example 1, and then the recombinant lentivirus was packaged to obtain the recombinant lentivirus before optimization, with a titer of 2.8×10 6pfu / mL, and then infected imDCs, and then induced and cultured for 36h to obtain mDC cells infected with the recombinant lentivirus before optimization. Western Blotting was used to detect the expression of PRRSV GP5 and Po-IFN-α in mDC cells infected with the recombinant lentivirus before optimization. The results showed that mDC cells infected with the recombinant lentivirus before optimization could highly express PRRSV GP5 and Po-IFN-α proteins (see Figure 3 ).

[0031] Example 2 Comparative experiment on the preventive effect of vaccines 18 piglets aged 6 weeks were tested negative for PRRSV by using ELISA kit and randomly divided into 3 groups, 6 piglets in each group. A booster immunization was performed on the 28th day after the first immunization.

[0032] Specific grouping: Experimental group A: injection of mDC cells infected with optimized recombinant lentivirus, 1000 cells / head / time; Experimental group B: injection of mDC cells infected with recombinant lentivirus before optimization, 1000 cells / head / time; Experimental group C: injection of empty-transfected mDC cells, 1000 cells / head / time.

[0033] On the 35th day after the first immunization, venous blood was collected, and the serum was separated to detect specific antibodies using ELISA. Peripheral blood lymphocytes were aseptically separated, and T cell subtypes were identified using flow cytometry. At the same time, a challenge experiment was conducted, and the challenge virus strain was PRRSV GD strain (2×10 5.5 TCID50 / mL, Gene Bank: EU825724.1, donated by Beijing Kangbaolihua Biotechnology Co., Ltd.), challenge dose: 10 3.5 TCID50 / mL, 2mL / head, nasal drops. After 14 days of observation, the clinical status of each group was analyzed, and venous blood was collected to separate serum and detect blood viral load using RT-PCR.

[0034] The results of the ELISA experiment are shown in Table 1. The results show that the S / P value of the specific antibody expression level in the serum of the piglets in the experimental group A was significantly higher than that in the experimental group B, indicating that after the nucleotide sequences of PRRSV GP5 and Po-IFN-α are optimized, the specific antibody expression level in the serum of the piglets can be increased, thereby improving the immune effect.

[0035] Table 1 ELISA test results on the 35th day after the first immunization

[0036] T cell subtypes were detected by flow cytometry, and the results are shown in Table 2. It can be seen that the proportion of CD3+CD4+T cells and CD3+CD8+T cells in experimental group A was significantly higher than that in experimental group B and experimental group C, indicating that the optimization of the nucleotide sequences of PRRSV GP5 and Po-IFN-α can improve the immune effect of mDC cells infected with recombinant lentivirus.

[0037] Table 2 T cell subsets results on day 35 after first immunization

[0038] Results of the virus challenge experiment: The piglets in experimental group C showed decreased appetite 3 days after the virus challenge, liked to gather and crawl together, and showed symptoms such as coughing, skin flushing, eating little or no food, and obvious weight loss 7 days after the virus challenge; the piglets in experimental groups A and B only had a temperature rise to 40.5 degrees 2 days after the virus challenge, and then the temperature gradually dropped, and no obvious clinical symptoms were shown. 14 days after the virus challenge, the RT-PCR method detected the virus load in the blood of the piglets in experimental group A (2.5×10 2 ±0.32) copies / μL, and the viral load in the blood of piglets in experimental group B was (5.6×10 4 ±0.29) copies / μL, and the viral load in the blood of piglets in experimental group C was (4.9×10 5 ±0.84) copies / μL. It can be seen that the optimization of the nucleotide sequences of PRRSV GP5 and Po-IFN-α can reduce the viral load in the blood of piglets.

[0039] Example 3 Comparative experiment on the therapeutic effect of vaccines 54 piglets weighing less than 20 catties that naturally fell ill in the farm (ELISA test confirmed porcine reproductive and respiratory syndrome) were selected and divided into 3 groups on average.

[0040] Experimental group A: injection of mDC cells infected with optimized recombinant lentivirus, 1000 cells / head / time; Experimental group B: injection of mDC cells infected with recombinant lentivirus before optimization, 1000 cells / head / time; Experimental group C: injection of empty-transfected mDC cells, 1000 cells / head / time.

[0041] After 24 hours, the same dose was injected again; the cell injection site was the muscle behind the piglet's ear.

[0042] The sick piglets showed symptoms such as dyspnea, anorexia, fever, eyelid edema, sneezing, purple ears and cyanosis of the skin on the extremities of the body. On the 4th day after treatment, if the above symptoms significantly subsided or disappeared and there was no mortality, they were judged to be cured, otherwise it was judged to be ineffective.

[0043] The clinical symptoms of 16 pigs in experimental group A disappeared, with a cure rate of 88.89%; the clinical symptoms of 13 pigs in experimental group B disappeared, with a cure rate of 72.22%; the clinical symptoms of 5 pigs in experimental group C disappeared, with a cure rate of 27.78%. It can be seen that the cure rate of experimental group A is significantly higher than that of experimental groups B and C, so the optimized recombinant lentivirus-infected mDC cells prepared by the present invention have better therapeutic effects.

[0044] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A vaccine that highly expresses porcine interferon-α and PRRSV GP5 protein, characterized in that: The artificial nucleic acid sequence of the PRRSV GP5 protein is shown in SEQ ID NO.1, and the artificial nucleic acid sequence of porcine interferon alpha is shown in SEQ ID NO.

3.

2. A vaccine for highly expressing porcine interferon-α and PRRSV GP5 protein according to claim 1, characterized in that: The vaccine contains a target gene fragment, which is obtained by connecting PRRSV GP5 protein, self-cleaving polypeptide T2A and porcine alpha interferon in series.

3. A vaccine for highly expressing porcine interferon-α and PRRSV GP5 protein according to claim 2, characterized in that: The artificial nucleic acid sequence of the self-cleaving polypeptide T2A is shown in SEQ ID NO.

2.

4. A vaccine for highly expressing porcine interferon-α and PRRSV GP5 protein according to claim 2, characterized in that: The vaccine is a mature DC cell containing a target gene fragment.

5. A vaccine for highly expressing porcine interferon-α and PRRSV GP5 protein according to claim 2, characterized in that: The vaccine is mature DC cells infected with a recombinant lentivirus containing a target gene fragment.

6. The method for preparing the vaccine according to any one of claims 1 to 5, characterized in that: The target gene fragment is inserted into the lentiviral expression vector to construct the target expression plasmid, which is packaged with the lentivirus and then infected with immature DC cells, which are then induced and cultured to obtain mature DC cells.

7. Use of the vaccine according to any one of claims 1 to 5 in the preparation of a medicament for preventing or treating porcine reproductive and respiratory syndrome.

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