Prokaryotic Expression Of Partial Gene Of P1Cap And The Establishment Of Indirect Elisa And Application For The Detection Of P1

Porcine circovirus belongs to Circoviridae and Circovirus.it is the representative species of Circoviridae,which includes two serotypes:PCV1and PCV2.PCV2can lead to many diseases and cause severe immunosuppression on pigs,and therefore easily to occur other disease secondarily or concurrently,this causes serious economic lose of pig industry.We got two strains of Procine Circovirus Type2-like factor (we call them P1and P2temporarily)in the serums of PMWS pigs in the diagnosis of PCV2in our lab. P1have648nucleotides and partially similar to PCV2. Although there was lots of progress on the research of the biological characteristics of P1, the epidemic of the virus is still unclear.So the preparation of pCap protein and the establishment of indirect ELISA based on the protien is very important for investigation and clinical diagnosis of P1.1. Expression and optimization the expression of3pCap tandem protein of P1in prokarvotic cellsThe371th nucleotide of P1had losted an A base compare with the459th nucleotide of reverse complementary sequence of PCV2,this was found by the analysis of sequence alignmen.The gene researched in this study is the sequence between370th and495th of P1. The primer was designed and the gene sequence was amplified three times,each sequence have special enzyme cut sites.After polymerase chain reaction,the sequences were cloned to the vector of pET-28a(+),and then the restructured plasmid was transformed into E.coli BL21strain (DE3).After the adding of IPTG, the recombinant protine which was14.7kDa was expressed. The best induction conditions were:cultivate the bacteria to the OD value between0.4and0.6in37℃,and then add IPTG to1.0mM,the expression of protine was maximum after induced for8h. Western blotting indicated that the protine induced could react with the rabbit anti-P1hyper-immuneserum. The result stated clearly the3pCap tandem protine was expressed successfully.2. The establishment of indirect ELISA based on the3pCap tandem protineThe ELISA plate was coated with3pCap tandem protine,after the optimization of conditions,the best reaction conditions was confirmed. The best antigen coating concentration was2.62μg/mL;the best serum dilution degree wasl:100; the best antigen coating time was coated in4℃overnight; the best blocking condition was blocking in37℃for1h with PBST which contained1%BSA;the best HRP antibody dilution degree was1:5000;the reaction between enzyme and its substrate last for four minutes. The stability,sensitivity,specificity and repeatability of this indirect ELISA were evaluated.The result of this study indicated that the indirect ELISA established have no cross reaction with the positive serum of CSFV, PRV, FMDV, PRRSV, PPV, SS2, PCV2, MHP, HPS and PCP.The variant coefficient of the indirect ELISA in a batch and between batches varied from1.50%to3.98%and2.91%to7.61%.respectively. The result of indirect ELISA was compared with IFA,the coincidence was94.1%.the coated ELISA panels was stable at-20℃for at least11months.The result of this study indicated that the indirect ELISA established in this study was specific and sensitive.We conclude,therefore,that indirct ELISA performed with3pCap tandem protien laid the foundation of the development of ELISA antibody test kit for P1.3. Epidemiological investigation of P1in Jangsu and four other provincesWe have used the established indirect ELISA to investigate1135serum samples,the samples were got from Jiangsu,Anhui,Shandong,Zhejiang and Shanghai five provinces,and the sera were collected between2008and2011.The two pig farms(Nanjing,Huaian) from Jiangsu province have156serum samples and the total positive rate was34.6%. The three pig farms(Huainan,Hefei,Chuzhou) from Anhui province have212serum samples and the total positive rate was18.9%. The two pig farms(Qingdao,Linyi) from Shandong province have148serum samples and the total positive rate was45.3%.The pig farm from Shanghai have90serum samples and the positive rate was14.4%. The three pig farms(Jinhua,Huzhou,Hangzhou) from Zhejiang province have529serum samples and the total positive rate was33.6%.405serum samples from pig farm of Hangzhou was investigated,the positive rate was38.5%,and the positive rate of sow,pre-starter,nursery pig and grower-finisher were53.3%,48.6%,34.5%,18.3%and20%.