Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine

A subunit vaccine and porcine PRRS technology, which is applied in the field of porcine PRRS subunit vaccine and its preparation, can solve problems such as irritation, and achieve the effects of improving immunogenicity, good protection effect, and easier separation and purification

Active Publication Date: 2020-11-24

WUHAN KEQIAN BIOLOGY CO LTD

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Abstract

The invention relates to the fields of genetic engineering and veterinary biological pharmacy, and in particular discloses a subunit vaccine for porcine reproductive and respiratory syndrome as well as a preparation method and application of the subunit vaccine. The invention firstly provides a fusion protein, wherein an amino acid sequence of the fusion protein is as shown in SEQ ID No. 1; and then the invention provides the subunit vaccine for the porcine reproductive and respiratory syndrome, wherein the subunit vaccine comprises the fusion protein. The fusion protein in the subunit vaccinefor the porcine reproductive and respiratory syndrome is obtained by conducting fusion expression on an ORF5 full-length gene of a PRRSV NADC30-like strain, which is optimized by virtue of a codon, and a gene of pseudomonas aeruginosa, from which a Domain III structural Domain is removed, and by virtue of an insect baculovirus/ insect cell expression system. The vaccine provided by the inventionis good in protective effect, capable of inducing effective humoral immunity and cellular immunity and is suitable for clinical prevention of the reproductive and respiratory syndrome.

Application Domain

SsRNA viruses positive-senseVirus peptides +15

Technology Topic

Genetic engineeringCellular immunity +12

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Examples

Experimental program(3)

Example Embodiment

[0039] Example 1

[0040] First, the construction of the destination protein expression vector

[0041] 1. Get the destination protein sequence:

[0042]PRRSV NADC30-like strains obtained from the NCBI gene sequence (Accession: MH651743), protein GP5 nucleotide sequence selected and optimized; Pseudomonas aeruginosa obtained gene sequence (GenBank: CP039293) from NCBI, outside the selected toxin PEA translocation domain (which removed DomainⅢ, hereinafter referred to as PEA △ DⅢ) sequences; the GP5 gene sequence and optimization of the PEA △ DⅢ series, and added 8 × his tag sequence, N and C-termini, respectively, together with NdeI and XhoI restriction site, biosynthetic recombinant protein (PEA △ DⅢ-GP5) gene sequence (as shown in SEQ ID No.1).

[0043] 2, the synthetic fusion protein pUC-PEA △ DⅢ-GP5 sequence with NdeI restriction sites XhoI and ligated into an expression vector pFastBacdual:

[0044] (1) was digested pUC-PEA △ DⅢ-GP5 and baculovirus expression vector pFastBacDual (available from biological Hui Feng, product number BR248):

[0045]

[0046] With ddH 2 O made up to 40ul.

[0047] Digestion conditions: 37 ℃ water bath was digested 1h.

[0048] Subjected to agarose gel electrophoresis after digestion, Gel Extraction, the steps follow standard procedures of gel extraction kit.

[0049] (2) PEA △ DⅢ-GP5 connected to pFastBacdual:

[0050]

[0051] Join conditions: 22 ℃ 2h.

[0052] Two recombinant baculoviruses rAc-PEA △ DⅢ-GP5

[0053] 1, the recombinant shuttle vector:

[0054] 4ul taken recombinant transfer plasmid pFastBacdual-PEA △ DⅢ-GP5 into 100ul competent E. coli DHlOBac (available from Gibco BRL). After the ice bath was 30min, 42 ℃ heat shock 1min, then the ice bath 3min, added 900ul non-resistant LB, 37 ℃ recovery 4h, LB third antibody coated plates (kanamycin, gentamicin, and tetracycline) and incubated 24-48h 37 ℃, positive colonies were screened by blue-white purified, recombinant bacmid extraction rAc- PEA △ DⅢ-GP5.

[0055] Extraction Method: Sterile White colonies were picked to a positive third antibody LB liquid medium and cultured 12-16 h, cells were collected with a 0.3mL solution I (50mmol / L glucose, 10mmol / L EDTA, 25mmol / L Tris-Cl ( pH 8.0)) resuspended, 0.3mL was added a solution of II (0.2mol / L NaOH, 1% SDS) slightly mixed, allowed to stand at room temperature 5min, was slowly added a solution of 0.3mL III (3mol / L CH 3 COOK, pH5.0) mixed ice bath 5-10min, 14000r / min centrifugal 10min, 0.5mL of isopropanol was added to the supernatant, mixed 5-10min ice bath, room temperature 14000r / min centrifugal 15min, 70% ethanol the precipitate was washed, dried and dissolved in 40μL sterile water and used immediately or stored at -20 ℃.

[0056] 2, recombinant baculovirus:

[0057] Using the lipofection method, the extracted recombinant shuttle vector was transfected into Sf9 insect cells (from Wuhan Branch bioprecursor Co.), and cultured at 27 ℃, after 48-72h cytopathic, i.e., the cell culture supernatants the recombinant baculovirus obtained using recombinant baculovirus were harvested immediately or placed in -80 ℃ stored.

[0058] Transfection method in accordance with the instructions liposomes (Lipo2000 available from Invitrogen).

[0059] Third, protein expression and purification purposes of PEA △ DⅢ-GP5

[0060] 1, the protein of PEA △ DⅢ-GP5 expression:

[0061] The harvested recombinant baculovirus seeded in suspension culture High Five insect cells TM (Commercially available from Invitrogen), the amount of bonding agent 0.01MOI, a cell density of 1.0 × 10 6 / Ml, cell volume of 400ml. At 3, 5, 8 days culture supernatant were harvested for protein expression by Western blotting PEA △ DⅢ-GP5, and the detection results are figure 1.

[0062] 2, the purified protein:

[0063] Nickel column purification methods using conventional affinity chromatography. Specific steps are as follows: HighFive 8 days after inoculation taken TM Cell cultures, centrifuged at 10000 rpm for removal of cells and cell debris, removed by 0.45um filtration membrane of small impurities; filtered supernatant was combined with a nickel column by column; heteroaryl wash buffer (50mM imidazole, 1 × PBS) by column wash heteroaryl; elution buffer (150 mM imidazole, 1 × PBS) was eluted through the column; eluent dialysis buffer (1 × PBS) dialyzed overnight at 4 ℃, protein was obtained. By SDS-PAGE and Western blotting after detection of protein purification. See detection results are figure 2 and image 3.

[0064] Preparing four, PEA △ DⅢ-GP5 subunit vaccine

[0065] After DⅢ-GP5 protein concentration was measured PEA △ obtained was purified by filtration with a sterile adjuvant ISA201 (commercially available from SEPPIC Co.) in a 1: 1 emulsion prepared in Comparative subunit vaccines into the PEA △ per milliliter vaccine DⅢ- GP5 antigen content of 200ug, stored at 4 ℃ stand. The preparation of sterile dialysis buffer (1 × PBS) with an equal volume of ISA201 adjuvant emulsified as a vaccine controls, at 4 ℃ refrigerator spare.

Example Embodiment

[0066] Experimental Example 1 PEA △ DⅢ-GP5 subunit vaccine safety test in mice

[0067] Optional 4 to 5-week-old Balb / C 10 mice were divided into 2 groups, 5 rats in each group, were immunized and control groups. Immunohistochemistry at day 0 and day 14, respectively, to the lower leg intramuscular injection in Example 1 was inoculated PEA △ DⅢ-GP5 subunit vaccine 200μL embodiment; control group in the same manner as the control vaccine injected volume. Observed for 14 consecutive days after immunization, mental test mice were normal appetite, good health and not have any adverse reactions, indicating that the good safety subunit vaccine in mice. Safety test results detailed in Table 1 mice.

[0068] Table 1

[0069]

Example Embodiment

[0070] Experimental Example 2 PEA △ DⅢ-GP5 piglets subunit vaccine safety and efficacy testing

[0071] 1, PEA △ DⅢ-GP5 subunit vaccine safety and efficacy tests on pigs:

[0072] 45-day-old piglets PRRSV negative selection 10, divided into two groups, immunohistochemical 5, 5 in the control group. PEA △ DⅢ-GP5 1.0ml subunit vaccine intramuscularly in Example 1 were obtained by immunohistochemical ears at day 0 and day 14, the blank control group was not immunized. Continuous observation for 14 days, pigs does not appear any clinical symptoms, the spirit, loss of appetite, body temperature were normal, it did not produce any adverse results show that the subunit vaccine for pigs security is good.

[0073] DETAILED piglets safety test results in Table 2.

[0074] Table 2

[0075]

[0076] 2, immune efficacy test PEA △ DⅢ-GP5 subunit vaccine

[0077] 45-day-old piglets PRRSV negative selection 10, divided into two groups. Respectively PEA △ DⅢ-GP5 subunit vaccine groups 5; 5 blank control group. Intramuscular vaccine 1.0ml, blank control group were not immunized Immunohistochemistry ears at day 0 and day 14. Respectively, in the second day of immunization (preimmune), after the second immunization blood 14,28,42,56,60 days before vena cava, the ELISA antibody levels detected; and detects IFN-γ serum on day 14 level (detection kit was purchased from BD Biosciences, BD-EL-P0003, according to the kit instructions) and neutralizing antibody titers. The test results are shown in Table 3, Figure 4 (Pig serum IFN-γ levels of the detection results of FIG.) And Table 4. The results show, PEA △ DⅢ-GP5 subunit vaccine with good immunogenicity, capable of stimulating the body to produce higher levels of specific antibody.

[0078] Piglets effectiveness assay (ELISA antibody levels detected) specific results are shown in Table 3.

[0079] table 3

[0080]

[0081]

[0082] ELISA detection experiment of the present embodiment employs antibody levels available from Wuhan Branch porcine reproductive and respiratory syndrome virus before Bio Co. of ELISA kit specific method:

[0083] Coating with dilutions of recombinant PEA △ DⅢ-GP5 protein to 1μg / ml, coated 96-well Elisa plates, 100μl / hole, 4 ℃ coated overnight. Add 200 ul of PBS washing solution, washing was repeated twice; The diluted (according to kit instructions, with serum diluent in the kit was diluted 1:40) test sera, positive (PRRSV specific serum, available from China Veterinary Drug control) 100μl from each plate was added to the antigen-coated wells, the positive and negative controls, each with two holes. Gently mix uniform vibration, incubated for 30 min counter 37 ℃. Add 200 ul of PBS washing solution, washing was repeated 5 times, and finally pat dry; per well HRP-labeled swine anti-lgG second antibody (diluted 1000-fold with PBS) lOOul, incubated for 30 min counter 37 ℃. Add 200 ul of PBS washing solution, washing was repeated 5 times, and finally tapped dry. Each well before adding substrate solution A 50μl, together with substrate solution B 50μl, mixing the reaction plate gently shaken at room temperature for 10 minutes the dark color; 50 l stop solution to each well, mix gently shaken reaction plate; disposed ELISA measured wavelength of 630nm, were tested.

[0084] Neutralizing antibody titer assay procedure is as follows:

[0085] (1) serum from

[0086] Sterile serum to be tested inactivated in a water bath 56 ℃ 30min.

[0087] (2) serum dilution

[0088]Was added to each well in 96-well cell culture plates in 50μl DMEM serum-free medium, followed by a continuous series of dilutions of serum to be tested for, from 1: 2 to 1: 256 dilution for each of four replicates per well 50μl.

[0089] (3) virus neutralization

[0090] Diluted with serum-free DMEM medium has good measure TCID50 of PRRSV NADC30-like strain of virus solution 100μl solution containing 400 TCID50 of virus, the virus was added to the diluted serum dilution wells, 50 l per well, 37 ℃, containing 5% CO 2 The role of the incubator 1h.

[0091] (4) was added Marc-145 cells

[0092] After covered with a monolayer of Marc-145 cells were trypsinized, with DMEM medium (containing 4% newborn calf serum) for blowing, counted and the cell density adjusted with DMEM medium (containing 4% newborn calf serum) to 2.0 × 10 5 / Ml, and cell suspension adjusted neutralized and inoculated in the completed cell culture plate 96, 100μl per well set 37 ℃, containing 5% CO 2 The incubator.

[0093] (5) the cell control

[0094] Cell control holes 8 provided in the above-described 96-well cell culture plate, 100μl DMEM added to each well of serum-free medium.

[0095] (6) determination results

[0096] Daily observe and record the situation cytopathic (CPE), continuous observation of the 4th.

[0097] It should be no cytopathic cell controls, calculated according to Reed-Muench Method subject PRRSV serum neutralizing antibody titers. Test results in Table 4.

[0098] Table 4

[0099]

[0100] Pigs in Table 4 for the 14 days after the second immunization serum collected, and the test results obtained with PRRSV NADC30-like. Apparent therefrom, the vaccine of the present invention mean titer, immune effect over.

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Classification and recommendation of technical efficacy words

Simple purification process
Easy to separate and purify

Subunit vaccine for porcine reproductive and respiratory syndrome as well as preparation method and application of subunit vaccine

A subunit vaccine and porcine PRRS technology, which is applied in the field of porcine PRRS subunit vaccine and its preparation, can solve problems such as irritation, and achieve the effects of improving immunogenicity, good protection effect, and easier separation and purification

AI-Extracted Technical Summary

Problems solved by technology

Abstract

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Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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