Prokaryotic Expression Of Porcine CypA And Its Influence On TGEV Replication In Vitro

Virus infection and pathopoiesia can not be separated from the participation of host cell protein. The host cell proteins may inhibit virus replication, or even get rid of the virus infection during the viral infection. And the proteins may also be utilized, to facilitate the reproduction of the virus. Cyclophilin A(CypA) is a highly conservative molecular chaperone protein which is widely distributed in animals and microorganisms. Researches for many years have proved that mammal CypA was related to many diseases. This study was designed to explore the role of the porcine CypA in Transmissible gastroenteritis virus(TGEV) replication, the following tests were processed to study the influence of porcine CypA on TGEV replication in vitro.1. Isolation and identification of TGEV in Sichuan province and establishment of the TGEV fluorescence quantitative RT-PCR detectionA strain of virus (SC-T) was isolated from diarrhea piglets samples in Sichuan by cultivation in PK-15cells, and the isolated virus was proved to be TGEV through PCR detection, physical and chemical properties experiment and microneutralization test. A pair of fluorescent quantitative RT-PCR primers were designed by the S gene of TGEV, specificity, reproducibility, sensitivity test and standard curve were processed to establish the TGEV SYBR Green fluorescent quantitative RT-PCR detection method.The ideal standard curve can be got while the concentration of the standard plasmid in5.32x107to5.32x101copies/μL, the method have a good reproducibility and specificity, and the minimum detection concentration was5copies/μL.2. Cloning and prokaryotic expression of Porcine CypAA pair of CypA amplification primers were designed by porcine CypA cds sequence, CypA gene of Landrace、Duroc、Meishan pigs and other pig breeds were cloned, the molecular evolutionary genetics analysis showed that CypA gene of chosen pig breeds and PK-15cells were highly conservative, similarity among them all were100%, and shared similarity with those of human CypA, mouse CypA and cat CypA were94.3%、92.1and83.6%, respectively.Porcine CypA gene was cloned into pET30a prokaryotic expression plasmid, named as pET30-CypA, the Recombinant plasmid was transformed into the competent cells of E. coli Rosstta after sequence analysis, and the expression conditions were Optimized. The expressed protein was pured by His tag fusion protein purification kit and identified by His tag Westernblot. Recombinant plasmid sequencing result was in line with expectation, the best expression condition of pET30-CypA was37℃0.2mmol/L IPTG induced recombinant bacteria to express4h. Recombinant protein was mainly existed in soluble expression and the puried protein was in highly purity and concentration. The His tag in the fusion protein can be detected by Wseternblot.3. The Influence of Porcine Cyclophilin A on TGEV Replication in VitroThe PK-15cell was cultured with different culture medium which contain different concentration of porcine CypA after infected with TGEV SC-T, the infected cells and supernatant were collected in different times postinfection. TGEV fluorescence quantitative RT-PCR was used to detect the vRNA contents of different samples, and the TGEV one-step growth curve of different groups were drew respectively.The intracellular and extracellular TGEV one-step growth curves were similar in different groups, At2-8hpi, TGEV vRNA increase rapidly in cells, at8-32hpi, vRNA maintained at a high level. TGEV vRNA in supernatant was in an s-shaped curve growth.0-6hpi was a period of incubation, vRNA stayed at a low level;6-28hpi was a stage of breakthrough, vRNA employed a logarithmic growth;28-32hpi, the growth rate slowed down, vRNA maintained at a high level and steped into stationary phase gradually. At0-8hpi, the Intracellular TGEV vRNA content of CypA groups were higher than control group, vRNA content was Positively correlated with CypA content. At8-32hpi, the Intracellular TGEV vRNA content was little difference in different group. At6-20hpi, all of CypA groups were got a higher extracellular vRNA levels than control group. The porcine CypA can promote TGEV replication in vitro.