The Prokaryotic Expression Of Tembusu Virus AH-F10 Strain E Gene And Establishment Of Fluorescence Quantitative RT-PCR Method

Tembusu virus disease is caused by the Tembusu virus,which can lead to a severe drop in egg production of the laying ducks and growth retardation.Duck and laying duck can be infected with the virus.It is characterized by its high prevalence morbidity and mortality.The incidence of the disease is up to 100%.And the mortality rate is in the 5%-10%.The infection has caused a serious economic loss to the poultry industry in China.The E protein of Tembusu virus contains the virus antigenic determinants,which plays an important role in absorpting virus and entrying into the host cells.The E protein is also the major antigen of flavivirus which can produce protrctive immune responses induced by neutralization pit.The gene segment E were successfully amplified from Tembusu Virus and were cloned into prokaryotic expression vector pET-32a(+)in this study.The recombinant fusion proteins His-E were highly expressed in E coli.BL 21 cell and the molecular weight of E protein was 54 kDa,which was in the forms of inclusion bodies.Western blot showed the recombinant protein could react with the porcine polyclonal antibodies against Tembusu Virus.It is showed that recominant proteins have good reactionogenicity.Recominant proteins were purified and the concentration is 2.978g/L,which is determination by UV absorption method.The purified E protein with Freund’s adjuvant emulsified volatile volume.Then immuning BALB/c mice every two weeks once according to each mice injected 0.2mg recombinant protein by subcutaneous.After the forth immunization,taking blood from eyeball.The IFA test was done by using the anti-E gene IgG as the primary antibody and the fluorescein isothiocyanated(FITC)conjugate goat anti-rabbit IgG as the second antibody,which demonstrated that the polyclonal antibody reacted specially with the E protein.In order to establish the method for detecting duck Tembusu virus by SYBR Green 1 absolute fluorescent PCR,one pair of primers were designed by primer 5.0 and synthesized for amplification of the E gene by RT-PCR in this study.The primers were abased on duck Tembusu virus E gene.The amplified products were identified and cloned into pMD19-T vector to construct the recombinant plasmid by transformation of E.coli DH5α,coating of Amp+ agar plates,the positive recombinant plasmid was identified by PCR and sequencing.Then the recombinant plasmid were extracted as the standard plasmid of fluorescent quantitative PCR.The standard plasmid was used as a quantitative template to establish the SYBR Green 1 real-time RT-PCR method for duck Tembusu virus,the specificity,sensitivity and repeatability of the real-time RT-PCR assay were evaluated respectively.The SYBR Green 1 real-time RT-PCR method for the duck Tembusu virus was successfully established by optimizing the reaction conditions.The results showed that the correlation coefficient of R2 was above 0.99,the mean repeatability tests coefficient of variation(CV)was 0.26%,and the sensitivity of the assay was 2×10~1copies/μL respectively.The 35 positive samples were detected with the total of 36 tissue samples from the ducks infected artificially with the duck Tembusu virus,the detection rate of 97%(35/36)was observed.While the detection rate of the conventional PCR was 42%.A SYBR Green 1 fluorescent quantitative PCR for detecting duck Tembusu virus was established.The method was more rapid,sensitive and accurate than conventional RT-PCR,and can be used to detect the clinical samples of duck Tembusu virus infection.