The Function Study Of Amino Acids In Loop DE Of Porcine Circovirus Type 2 Capsid

Porcine circovirus type 2 (PCV2) is closely associated with postweaning multisystemic wasting syndrome(PMWS),which cause severe diseases such as poor growth and dyspnea in piglets, porcine dermatitis, reproductive failure, etc. PMWS is endemic in the majority of the swine-producing countries, causing a significant economic losses in the swine industry worldwide.In this study, we compared the sequences of PCV1 and PCV2. Based on the wild type capsid protein of PCV2, we replace several amino acids in the loop DE which is different from PCV1, and constructed several recombinant plasmids and infectious clones of PCV2.Our study will help for future investigation on the production of VLPs vaccine and mechanism of virus pathogenesis. Our experiment was conducted as follows:1.Preparation of recombinant protein by Escherichia coli expression system. Two modified complete coding sequence of PCV2 ORF2 (open reading frame 2), which encodes Cap protein, were cloned into expression vector pET100, namely pET100-PCV2-M1(M1) and pET100-PCV2-M2(M2).Then the correct recombinant plasmids were transformed into BL21 competent cells and induced for protein expression. According to the results of SDS-PAGE and western blots, there was a significant band shows at expected position (about 26kD),indicating that the target gene could be expressed successfully in E.coli expression system.2.VLPs assembly and infection. The target proteins were purified and dialyzed into VLPs (virus-like particles). For morphological study, the rPCV2-VLPs were observed under TEM(transmission electron microscope) and shown a lots of particles with a diameter about 20nm.This particles have the same regular shapeand size compared with wild type PCV2, which means the purified protein expressed in E.coli could correctly self-assembled into VLPs.For Immunofluorescence assay (IFA), fluorescence signals were captured in PK-15 cells by using mouse PCV2-postitive serum as primary antibody. Besides, no specific fluorescence was observed in control cells. These results revealed that the two rPCV2-Cap-VLPs have ability to infect PK-15 cells and have good antigenicity and specificity against the PCV2 antibody.3.Construction of infectious clone and rescued virus. Two kinds of virus genomes were connected into pSP72 vector to construct double copies infectious clones, pSP72-PCV2-IF-DE-M1 (IF-M1) and pSP72-PCV2-IF-DE-M2(IF-M2),by using gene clone techniques. These two infectious clones were then transfected into PK-15 cells, and identified by TEM and qPCR. All theseresults showed that the two infectious clones could product rescued virus. Besides, the real-timequantitative PCR result showed that there was a significant difference betweenthesetwo infectious clones, the copy numbers of IF-M1 wereequal to PCV2, howeverthe copy numbersof IF-M2 weremuch fewer than PCV2.All above, the IF-M1(C-S) had no interruptionof virus replication but IF-M2(CS-RD)couldreduce virus replication in vitro.