Development And Application Of Protein Microarrays For Detecting Antibodies To Porcine Circovirus TypeⅡ Using Recombinant CAP Protein

Porcine circovirus(PCV)is a peplosless single minus strand DNA virus. It is the smallest virus that ever found. PCV is classified into PCVI and PCVII by its antigenicity and genome. There are a lot of laboratory diagnostic methods for virus antigen and specific antibody of PCVII, such as viral isolation, PCR, EMC and ELISA.Protein Chip, so called Protein Microarray, is a kind of new technique that can analysis many targets of creature simple at the same time. It can not only retain the character of antigen- antibody response but also have the feature of high-flux, low cost, high-parallel, miniaturisation and steep response that chip has. It has become the new direction of animal loimia diagnose.In this study, visual recombinant protein microassay and FITC wink protein microassay of PCV II were made and optimized. They could detect the antibody of PCV II in swine serum sensitively. The CAP was purified and concentrated, which expressed by prokaryotic expression system. It was printed onto the chip as capture antigen to capture the PCV II specific antibody. The processes inculuded:(1) The preparation of protein antigenPCVII CAP gene was amplified by PCR, the PCR product was cloned into pMD-18 vector, and then convert into E.coli JM109. the plasmids and the vector pET30a were cut by nucleotide restriction enzyme and then linked by T4 ligase. The recombinant plasmids were checked by PCR, nuleotide restriction enzyme cut reaction and sequencing. The positive recombinant plasmids were transformed into BL21 (DE3) and induced by IPTG. Use ultrasonication and other chemical lysis reagent to purify the inclusion body primarily. Later, the protein was further purified by Ni-NTA affinity chromatography method. Finally recombinant proteins were dialysised to refold. Western blotting showed that the recombinant proteins had good antigenicity.(2) the visual protein microarray and optimization of conditionsSpotting the capture antigen onto the chip substrate which was chemically modified with amino-; and the condition of chip preparation, capture antigen concentration, and the percentage of developer were optimized. Finally, the chip's linear detection region and detection interval were checked by the detcetion of 60 blood-serum samples, and the consequence was comparaed with the detection of using ELISA kit.(3) The FITC microarray and optimization of conditionsSpotting the capture antigen onto the chip substrate which was chemically modified with amino-; and the condition of chip preparation, capture antigen concentration, and the percentage of developer were optimized. Finally, the chip's linear detection region and detection interval were checked by the detcetion of 60 blood-serum samples, and the consequence was comparaed with the detection of using ELISA kit.(4)In this study, recombination protein CAP was obtained by prokaryotic expression. Because the condition of purification and refloding were co-optimized, the efficiency of protein purification and renaturation was raised and cost was saved. And Primary results showed that our home-build visual protein micorassay and FITC protein microassay had good feature morphology and high reproducibility. Besides the detetion range of recombinant protein microarray were suprior to the indirect ELISA which was set up with the same capture antigen, and the sensitivity of microarray was 2 times more than indirect ELISA. The progress of this research laid solid foundation for the setup of higher density protein microarray.