Expression And Potential Application Research On ORF2 And ORF3 Gene Of Porcine Circovirus Type Ⅱ

Postweaning multisystemic wasting syndrome(PMWS)is a new emerging disease with symptoms of weight loss,dyspnoea,diarrhoea,jaundice,interstitial pneumonia, pallor,ulceration,and enlarged lymph nodes.Histopathological lesions are characterized by lymphocyte depletion and histiocytic infiltration of lymphoid tissues. The disease can result in significant economic losses to the swine industry.Porcine Circovirus 2(PCV2)is the etiological agent of PMWS.Moreover,PCV2 is also associated with many other diseases such as respiratory disease,reproductive failure, porcine dermatopathy and nephropathy syndrome(PDNS),and hepatitis.Diseases associated with porcine circovirus type 2(PCV2)infections are becoming a major problem for the swine industry worldwide.In this study,ORF2 and ORF3 gene of PCV2 were cloned and expressed to develop an effective live vaccine against PCV2 infection and explore the function of ORF3 protein to T lymphocytes of mice and cancer cells(HT 29 and SGC 7901).Whole ORF2 genes of six positive clinical PCV samples from six areas of Shandong province were amplified by PCR and sequenced.Comparison of the orf2 gene sequence with the sequences in databases reveals a high degree of conservation. Among the six orf2 genes,nucleotide identity is 93.3 to 99.7%,amino acid identity is 92.3 to 100%.The amino acid sequence alignment of the capsid protein encoded by orf2 gene showed these six PCV2 strains exhibited two major regions of greater heterogeneity at residues 57～90 and 190～220.Phylogenetic analysis with nine isolates reported in GenBank show that the PCV2 strains were subdivided into two clusters.SDâ… ,SDâ…¢,SDâ…¤,SDâ…¥share close genetic relationship with strains from France(FRA)and China(ND).They probably have the same ancestor.SDâ…¡and SDâ…£form a larger cluster with other seven isolates.They share the closest genetic relationship with strains from China(HN).Restriction fragment length polymorphism (RFLP)analysis with orf2 genes of other 13 isolates of Shandong reported in GenBank show that they could be divided into 5 diferent genotypes CHN-2H, CHN-2F,CHN-2I,and two new emerged genotypes,named CHN-2J and CHN-2K. The CHN-2H was the dominant genotype of PCV2 among genotypes in Shandong Province.off2 gene of CHN-2H genotype should be chosen to develop vaccines against PCV2 infection.Lactic acid bacteria(LAB)are noninvasive,nonpathogenic,gram-positive bacteria and are considered as "generally regards as safe"(GRAS)strains and thus attractive tools to deliver antigens and therapeutic molecules at the mucosal level. orf2 gene encodes the nucleocapsid protein(Cap)of PCV2,which is an antigen important for both early diagnosis and development of vaccines.In this work, Lactococcus lactis was used as vehicle to deliver the PCV2 antigen in an attempt to develop oral vaccine against circovirus.An orf2 gene with a deleted nuclear localization signal sequence(dcap)was cloned into two E.coli/L,lactis shuttle vectors pSEC and pSEC:LEISS under the control of a nisin promoter.Intracellular or extracellular expression of the dCap was confirmed by western blot analysis.Results showed that dCap protein was expressed in the cytoplasm of two recombinant bacteria pSEC-dCap and pSEC:LEISS-dCap strains.Also dCap protein can be secreted into the medium in pSEC:LEISS-dCap strains.The culture condition and nisin induction were optimized for maximum dCap yield.Results indicated that strains were induced at OD600=1.0～1.2 with 10～12 ng ml^-1of nisin at 18℃for 6 hours is the optimal condition for dCap production.The yield of secreted dCap protein was achieved about 1.2 mg L^-1culture.The production of dCap was increased at least two-fold.Survive in the gastrointestinal environments is the key for recombinant L.lactis to play its role as vaccine.The survival properties of recombinant L.lactis pSEC: LEISS-dCap strain was studied in imitative gastrointestinal environments,such as artificial gastrofluid(pH=1.5～4.5),artificial intestinal fluid,bile(0.3～3.0g/L), and high concentration of NaCl(30～80 g/L).The results indicated that the recombinant strain could tolerate 6%NaCl solution and survived well in artificial gastric fluids at pH=1.5～4.5.After 3 h,the viable bacterial cells could reach about 10⁷CFU/ml.The recombinant strain could slowly grow in the artificial intestinal fluid. However,the recombinant strain could not tolerate bile above 0.1%concentration.So tolerance to bile is the bottleneck in practical application as oral live vaccine. We observed immungenocity of mice upon oral administration of strain cultures expressing the PCV2 antigen.The cultures have no effect on growth of mice. Significantly higher levels of PCV2-specific IgG in the sera of mice were induced than those of control mice(P＜0.02).Furthermore,the payer's patches in the intestines of mice of vaccinated group were more and prominant than those of control groups, which demonstrate that dCap protein was delivered to intestinal sites and induced mucosal immune response.This suggested that it is feasible to use L.lactis as an antigen delivery vehicle for developing oral vaccines against PCV2 infection.ORF3 is a newly identified protein from porcine circovirus 2(PCV2).The roles it plays have not been determined now.In this study,ORF3 gene was cloned and its sequence was analyzed.The native ORF3 contains a large number of E.coli rare codons(RC)including 5.7%AGG or AGA coding arginine and 4.8%CCC coding praline.We modified the 9 of the 11 E.coli RCs present in ORF3 with their optimal synonymous degenerate codons.The resulted mORF3 and the native ORF3(nORF3) were subcloned into the prokaryotic expression vector pET22b(+)in order to produce 6 His-tagged fusion protein in the BL21(DE3)cells.SDS-PAGE and Western blotting analysis demonstrated that mORF3 was capable of highly expressing in BL21 (DE3)than nORF3.The fusion protein amounted to 16%of the total bacterial proteins after induction with 0.1 mM IPTG for 4 h at 37℃.These will facilitate further functional study on property and antibody preparation of ORF3.To determine the role of the ORF3 protein in modulation of T-lymphocyte function of mice,eukaryotie expression system was constructed.The results demonstrated that animals of the ORF3-injecteded group showed significant upshifts of CD8⁺T-cell subsets and CD4⁺T-cell subsets of peripheral blood lymphocytes compared to the control mice(P＜0.05)at various time points after muscle injection.While,the proportions of the CD4⁺ and CD4⁺ CD8⁺ cells were still in normal range.These results showed that ORF3 can boost T-lymphocyte immune response.To determine whether ORF3 protein could trigger human cancer cell apoptosis, ORF3 genes were transiently expressed in colon cancer cell line(HT 29 cells)and gastric cancer cell line(SGC7901 cells)for apoptotic activity assay.After 48 hours posttransfection,conformations of cells and nucleolus appeared obviouse apoptosis feature;Flow Cytometry showed that G₀-G₁ phase fraction increased,while that of S phase decreased;LDH assay showed that there was no significant difference(P＞0.05) in LDH activity in the mediums between the controls and test groups,which proved that cells underwent apoptosis not necrosis.The results suggest that the ORF3 protein plays a major role in the induction apoptotic activity of infected cells.These will facilitate further study on apoptotic pathway and mechanism of ORF3.