| Although the great economic success in our nation has brought us a better life, we, unwillingly, would have to accept the austere situation that the incidence of sexually transmitted diseases (STD) has increased rapidly over the past decades. Condyloma acuminata (CA), which occurs frequently in our daily clinical practice, is a kind of STD caused by Human papillomavirus (HPV). Despite of the availability of several alternative therpies for CA, its easiness to relapse really frustrate us. Fortunately, lots of researches have revealed that deficiency of local or regional cell-mediated immune responses to HPV is probably one of the major mechanisms leading to the occurrence and insistence of CA. That is to say, regression of HPV-associated lesions might to some extent depends on activated cell-mediated immunity. Therefore, it might be an innovative way to prevent the recurrence of CA or even toeradicate HPV by strengthening the cell-mediated immune responses locally involve in.Research on Papillomaviral vaccine has progressed rapidly in recent years. It has been reported that vaccination with HPV16 E6- or E7- derived peptides increased antiviral and antitumor effects by inducing cell-mediated immune responses both in vitro and in vivo, for instance, the activation of antigen-specific cytotoxic lymphocytes. Researchers are looking forward to producing vaccines that could be used as a new approach to treat CA effectively. As we all know, CA is caused by HPV that consists of no less than about 100 different subtypes among which low-risk HPV subtypes such as HPV 6,11 have been considered to be major pathogens causing CA. However, until nowadays, seldom reseach that focuses on HPV 6 or 11 peptide-based vaccine has been conducted. In this project, by means of moleculr biology, we successfully cloned HPV6b E7 gene, constructed expression vector pGEX-4T-2/HPV6b E7, and gained GST-HPV6b E7 fusion protein in large amout. What we have done in our project not only has provided large quantities of HPV6b E7 protein necessary for advanced researches on its immunologic characteristics as well as its function, but also has prepared with preliminary experience for producing of HPV6b E7 peptide derived vaccine.Methods 1. Polymerase chain reaction (PCR) and subcloning: Primersdesigned with EcoR I, Xho I endonuclease sites were used in PCR to amplify HPV6bE7 gene from pUC19/HPV6b genome. Amplified fragment was subcloned into pGEM-T vector to construct subcloning plasmid named as pGEM-T/HPV6b E7 which then was identified by EcoR I and Xho I digestion as well as sequencing.2. Construction of prokaryotic expression vector: Having been digested by restriction enzymes EcoR I and Xho I, HPV6b E7 fragment and linearized expression vector pGEX-4T-2 were ligated to construct recombinant prokaryotic expression vector named as pGEX-4T-2/HPV6b E7 which then was identified by EcoR I and Xho I digestion as well as sequencing.3. Induction of GST-HPV6b E7 fusion protein: A single colony of E.coli P2392 cells transfected with pGEX-4T-2/HPV6b E7 was picked and inoculated LB medium. And growed liquid cultures with vigorous shaking over night, then added IPTG to induce fusion protein expression. Subsequently, collected the liquid cultures, lysed the cells using a sonicator, and centrifuged to separate supernatant and pellet. At last, analysized the products by SDS-PAGE.4. Purification of GST-HPV6b E7 fusion protein: Solubility of the fusion protein was analysized, for whether the fusion protein is soluble determined the method of purification that would be adopted. Added 50% slurry of Glutathione Sepharose 4B to purify the fusion protein and measured the concentration offusion protein by method of Folin-Phenol.ResultsWe have succeeded in constructing the subcloning plasmid pGEM-T/HPV6b E7 and recombinant prokaryotic expression vector pGEX-4T-2/HPV6b E7. Both of them were identified by restriction enzyme digestion and sequencing, which showed that HPV6b E7 gene had been inserted into the multiple clo...
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