High Expression, Purification And Identification Of PBV220-HPVL1 And PBV220-HSVgD

ObjectiveIt has been well known that HPV infection is the main etiologic factor for invasive and pre-invasive cervical neoplasia.HPV DNA has been found in more than 90% of cervical cancers, and HPV16 represents about 50% of the viral types identified.However, women infected with high-risk types of HPV, only a small subset will develop invasive cervical cancer, suggesting that other cofactors must be present for the development of malignancy.HSV-Ⅱinfection may act in conjunction with HPV infection to increase the risk of invasive cervical cancer.L1 gene was got from HPV by PCR and gD gene was amplified from HSV strain by means of PCR and combinated with pBV220,which is prokaryocyte expression plasmid to build the constructs of pBV220-L1 and pBV220-gD.In order to induce pBV220-HPVL1 and pBV220-HSVgD expression in E.coli DH5αand to purify the recombinant proteins to obtain large amount of proteins of pBV220-HPVL1 and pBV220-HSVgD and to identify them,and provide an important experimental materials for the study on prophylactic vaccine of cancer of the cervix.MethodThe recombinant plasmids were transformed to E.coli DH5αand pBV220-HPVL1 and pBV220-HSVgD expression in the form of inclusion body were gained after induction with temperature. Inclusion bodies collected after the breakage of bacteria through sonication were subjected to washing with 2 mol/L urea. Then the rabbits were immunized with purifing inclusion bodies and inclusion bodies solubilized in the presence of 8 mol/L urea by subcutaneous injection in the test group, PBS solutions were injected in the control group by subcutaneous injection. The second immunization was completed after 2w. After that, the rabbits were immunized every one week for 6 times.Blood was collected from the rabbit at 5 days after the seventh immunication and the titers of HPVL1 and HSVgD antibodies were determined by agarose double immunodiffusion and the interest proteins of pBV220-HPVL1 and pBV220-HSVgD were verified by Western blot.Result1,The recombinant plasmids pBV220-HPVL1 and pBV220-HSVgD were transferred into E.coli DH5α.After 42℃induction for 6h and 5h,respectively,pBV220-HPVL1 and pBV220-HSVgD recombinant proteins were successfully expressed in E.coli DH5αand could be detected by SDS-PAGE, which exist as inclusion bodies principally. The molecule weights of pBV220-HPVL1 and pBV220-HSVgD recombinant proteins were about 64KD and 13KD respectively.2,The highly purified proteins of HPVL1 and HSVgD were obtained through separation and purification and their concentrations were 5.9mg/ml and 4.6mg/ml respectively,which were tested by Brand ford.3,Polyclonal antibodies against HPVL1 and HSVgD were generated by immunizing rabbits with purified pBV220-HPVL1 and pBV220-HSVgD recombinant proteins and their titers were detected by agarose double immunodiffusion with recombinant protein as antigen ,the results showed that the titer of antiserum against HPVL1 or HSVgD was about 1:16,and a single precipitation band was formed between antiserum against HPVL1 or HSVgD and immunogen and the result also showed that polyclonal antibodies against HPVL1 and HSVgD satisfactoried in purity.4,Western blotting indicated that the antiserum against HPVL1 or HSVgD could bind with the expressed recombinant protein specifically and the result showed the expressed proteins were the pBV220-HPVL1 and pBV220-HSVgD recombinant proteins.ConclutionThe recombinant proteins of pBV220-HPVL1 and pBV220-HSVgD were highly expressed in E.coli DH5αand the prepared two polyclonal antibodies,which anti-HPVL1 and anti-HSVgD ,were highly specific and their high titers could be used in further experiments.