Study Of The Porcine Parvovirus Nonstructural Protein NS1 And Structural Protein VP2

Porcine Parvovirus (PPV) infection is of economic concern to pig breeder worldwide. It is a major cause of stillbirth, mummification, embryonic death, infertility (SMEDI) syndrome, and sometimes, it is the cause of diarrhea and dermatitis. Co-infection of PPV and porcine circovirus 2 (PCV2) has been shown to be responsible for a significant portion of field cases of porcine postweaning mulisystemic wasting syndrome (PMWS). Some types of inactivated PPV vaccines are available. Vaccination with inactivated PPV is generally considered to offer good protection against PPV infections and is used to control reproductive failure due to PPV. In farms where vaccination is carried out, the majority of susceptible pigs should be seropositive. So, the pathogen detection method and differentiating serological method which can distinguish vaccinated pigs from naturally-infected ones, are necessary.Non-structural polyprotein 1 (NS1) (86 kDa) is the major nonstructural (NS) protein produced during PPV infection. It was reported to be a phosphorylated polypeptide, and be highly conserved among the parvoviruses. It is produced during the early phase of replication, before the onset of viral structural protein synthesis. Among the parvoviruses studies, NSl protein of Minute Virus of Mice (MVM) and the Aleutian Mink Disease Parvovirus (ADV) had been reported to have important functions during infection in vitro. Detection of Parvovirus B19 NSl specific antibody is useful in determing the level of protection from a capsid-based vaccine, as the emergence of anti-NS1 antibodies indicates parvovirus infection. Since the inactivated PPV vaccines that consist of semi-purified, chemically inactivated virus can elicit only antibodies against the structural proteins, assays which specifically detect antibodies against the NSl protein may be useful in differentiating vaccinated animals from infected ones. The possibility of suppression of porcine parvovirus (PPV) reproduction in the culture of thyroid gland cells of a swine that contain the integrated genes for asRNA against the nonstructural proteins of the virus has been studied. 10 cell lines with the antisense RNA genes have been obtained. The line with the maximal number of integrated gene copies was used to inflict with the parvovirus. The expression of antisense RNA in this cell line was shown to lead to 95% suppression of PPV replication as compared with the control cell line. That is to say, suppression of replication of swine parvoviral antisense RNA against thePPV ns gene in swine thyroid gland cells.RNA interference (RNAi) refers to the inhibition of gene expression by dsRNA. dsRNA can not only take part in gene regulation, but also inhibit gene expression of pathogenic microbes. RNAi is believed to function as a defense against viruses. In the medicine study, this technique may be the star in gene therapy of tumor, infections and inherited disease. Inhibition of virus replication using specific RNAi has been reported for many pathogens. It makes possible to develop RNAi based antiviral therapeutics.The particle of PPV is composed of three structural proteins: VP1, VP2 and VP3, of which VP2 is the major component of virus capsid. The structure of VP2 consists of an eight-stranded anti-parallel β-barrel motif with four large insertions between the β-strands called loops, which make up much of the surface of the virus capsid. These loops contain many B-cell epitopes, being the immunodominant domains of the PPV capsid. Other antigenic regions are also found at the N- and C-termini of VP2. It has been demonstrated that VP2 induces neutralizing antibodies and can protect pigs against virus challenge, indicating that VP2 is a potential candidate of a subunit vaccine. The recombinant VP2 of PPV using a bacculovirus expression system can self-assembles into empty PPV virus-like particles (VLPs), which have been shown to be highly immunogenic. PPV-VLPs had been engineered to express foreign polypeptides, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses.Based on the above mentioned research background, the present study investigated the application of the PPV ns1 and vp2 gene in prevention and therapy of the disease, which summarized below:1. An enzyme-linked immunosorbenr assay (PPV-whole virus-ELISA) was developed for detection of antibodies against PPV in swine sera. The antigen (PPV) used for the assay was purified and condensed after implanted in IBRS-2 cells. Through checkerboard matrix titration, the optimal amount of antigen coating ELISA plate was determined to be 300 ng/well. A dilution of 1:80 was selected as the best dilution to discriminate positive from negative sera with minimized false positive or false negative results. The difference between PPV-whole virus-ELISA and LAT was compared. Detecting 47 infected serums, the consistency rate between them reaches 93.3%, while detecting 61 immunized serums, the positive rate of PPV-ELISA is remarkably higher than LAT (P<0.01). In addition, PPV-ELISA have a strong sensitivity, high specificity, high accuracy, and is very smart, so it can be fully used to detect the level of antibody and the large account of serums in one time. Finally, the diagnosis was carried out to detected a large amount of serums in Central of China, the result shows that the incidence of PPV is high, especially in gilts and sows, it is are higher than 90%.2. A pair of primers was derived from the DNA sequences that have been published. The PCR method was developed for the detection of PPV nsl gene. The results revealed that the expected 1,100 bp fragment could be amplified from many kinds of tissues of infected foetus and passaged cells. This method could detect at least the virus of 1 TCID50.7 positive of 8 samples from clinical sick swine were detected by PCR and only 6 positive were detected by HA test. These results showed that the PCR method had the specificity and high sensitivity.3. The complete nsl gene of PPV was amplified from the replicative form DNA of the PPV China strain by PCR using two synthetic oligonucleotide primers. The nsl DNA was purified and ligated into pMD-T vector. After transforming E. coli DH5a, ampicillin resistant colonies were isolated and plasmid DNA was prepared. The recombinant plasmid containing the nsl was named as pMD-ns1. The plasmid was digested and ligated into the corresponding sites in expression vector pET-17b. The recombinant expression plasmid pET-ns1 containing the nsl gene was identified by restriction analysis and sequencing.The nsl expression plasmid pET-ns1 was transformed into E. coli BL21 (DE3). Recombinant strain BL21 (DE3)/pET-ns1 were induced by 1 mM isopropyl-β-D-thiogalactopyranpside (IPTG) for expression of the NSl protein. The inclusion bodies were obtained and the rNS1 protein was purified under denaturing conditions. The purified and refolded proteins were analyzed with protein assay system and SDS-PAGE to determine the protein concentration and yield. The purity was quantified and stored at -20℃ with 50% glycerol.The result indicated the full-length nsl gene (1,989 bp) was cloned from PPV viral DNA using PCR. SDS-PAGE and Western blotting identified the expression of NSl protein in E.coli. Compared with vector transformed control bacterial cells, the pET-ns1 transformed bacteria showed three additional bands. A band at approximately 86 KDa position is likely to be NSl protein according to literature report, the 86 kDa band reacted strongly with anti-PPV positive serum in Western blotting. The recombinant NS1 protein was harvested at about 20 mg - 40 mg per liter culture.4. To differentiate pigs infected with PPV from those vaccinated with inactivated whole virus vaccine, an ELISA based on detection of a non-structural polyprotein 1 (NSl) was developed. A threshold of 0.23 optical density units was established and the assay had high specificity (100), sensitivity (88), accuracy (90) and positive predictive value (100) using HI as standard method. Reproducibility test revealed that the coefficients of variation of sera within-plates and between-run were less than 10%. The assay showed no cross-reactivity with antibodies to PRRSV, PRV, FMDV, APP, toxoplama or chlamydia. Sera obtained from pigs infected with PPV reacted with recombinant NS1protein in the EUSA. Sera from pigs vaccinated with inactivated whole virus did not recognize this protein in the ELISA. In contrast, antibodies against PPV whole virus was present in both PPV infected and vaccinated animals. In conclusion, the PPV-NS1-ELISA could differentiate PPV infected vs. inactivated PPV vaccinated pigs and be applied in disease diagnosis and surveillance.5. Pichia pastoris have the characteristic of efficiently secreting foreign proteins but little self-protein and can perfect post-translation modification. For the sake of better eliminating non-specific reaction and enhancing the immunogenicity of PPV NS1 protein, excretion expression of PPV NSl protein in Pichia pastoris yeast has tested. Recombination expression vector pPICZaB-ns1 with single copy of target gene constructed. Pichia pastoris strains GS115, SMD1168H were transformed by these recombianats. The X-33/pPICZaB-ns1, GS115/ pPICZaB-ns1, SMD1168/pPICZaB-ns1, were obtahed. The expression was induced by methanol. SDS-PAGE, Wetern-blot, was performed to confirm the expression of PPV nsl gene. The result showed the products were too low to application.6. To identify the siRNA interference ability for the replication of PPV, based on thesequence of PPV nsl gene, two DNA sequence (named "ns-244" and "n5-254")containing small bairpin structure were designed and synthesized. The complement formwas obtained by annealing and inserted into vector pSilencerTM4.1-CMV neo, and therecombinant plasmid (pSilencer-ns244 and pSilencer-ns254) was transformed intoE.coli DH5a strain. Finally the plasmid identified by restriction enzyme was used forsequence analysis. The results indicated the recombinant was cloned and the aimsequence was obtained. Meanwhile, in order to obtain the PPV NS-mut4GFP fusionprotein, the completed PPVns1 was cloned into the eukaryotic expression vector ofpCM4, resulting recombinant expression plasmid pcM4 — ns1. The plasmidpSilencer-ns244 (or pSilencer-ns254) and plasmid pcM4—ns1 were cotransfected intothe PK-15 cells by transfection reagent (Lipfectin 2000), then the numbers of fluorscentcell were measued after 24h and 48h with by flow cytometric. The results indicated thatthe expression of the PPV NSl protein was suppressed. On the other hand, the purifiedpSilencer-ns244 (or pSilencer-ns254) was transfected into culture PK-15 cells when cellmonolayer was at about 50%-70% confluency. High multiplicity of infection PPV wasadded 6h after transfection, and inhibition of virus replication was detected 24h and 48hafter virus inoculation by detection the TCID50 and CPE. The results showed thatpSilencer-ns244 and pSilencer-ns254 had induced inhibition of viral replication inPK-15 and decreased the cell death.7. First, an Xho I site was introduced into the N terminus of PPV vp2 by site-directed mutagenesis using PCR. The unique Xho I cloning site adjacent to theorginal ATG of PPV vp2 . Then, the PPV vp2 was cloned into pMD18-T to constructed the pMD-vp2. In order to construct a recombinant baculovirus expressing TVP2, oligonucleotide 5'-ttt gga tcc acc atg gcc aga cac aag att gtg gca ccc gca aaa cag ctt ctg etc gag-3' and its complementary were designed to encode the FMDV T cell epitope of residues 200-213 of VP1 plus an initiation codon and BamH I site in N terminus, Xho I site in C terminus. The oligonucleotides were synthesized to regenerate by PCR. For PCR, 800 ng of each oligonucleotide was denatured for 5 min at 95 ℃. The samples were then subjected to 30 cycles of amplification under the following conditions:95 ℃ for 1 min, annealing at 55 ℃ for 1 min, and extension at 72 ℃ for 2 min. A final polishing step was carried out at 72 ℃ for 7 min. The reactions were done with Taq DNA Polymerase. The PCR product was treated with phenolchloroform and precipitated with ethanol. The PCR product was digested with BamH I /Xho I and subcloned into BamH I /Xho I - digested pMD-vp2 to construct the plasmid pMD-Tvp2. The plasmid identified by restriction enzyme. The mutant geneses containing insertions can be rescued from the respective plasmid by BamH I / Sal I digestion, gel-purified, and suncloned into BamH I /Sal I pFastBacTM 1 baculovirus transfer vector. Recombinants pFast-Tvp2 should be analyzed by restriction analysis of minipreps and clones containing the right insert in the correct orientation confirmed by sequencing across the BamH I insertion site. This sequencing step of the the insertion regions, as ligation reactions with the large-size baculovirus transfer vectors are susceptible to the introduction of mistakes in sequence. The next step involves the transfection of insect cells (Spodoptera frugiperda: Sf9) by the Lipfectin 2000. After 4-6 days, when Sf9 cell start to show cyteopathic effect (CPE), culture supernatant are plated for baculovirus isolation in the presence of X-Gal by their LacZ-negative phenotypes, white plaque are recoved, and the recombinant baculovirus are plaque-purified, until high titer virus stocks are obtained. When the recombinant baculovirus AcNPV-TVP2 have been obtained, they are used to overeapress large amounts of the protein. The assembly of TVP2 had been assessed by SDS-PAGE and western blot anaysis, hemagglutination anysis, and elestron microscopy.