Gene Cloning And Expression Of Human Papillomavirus Type 6 Capsid Protein L1 And L2 And Immunological Analysis Of The Assembled Virus-Like Particles

Human papillomaviruses (HPV), infecting mainly human cutaneous and mucosa in a strict species- and tissue-specific manner and causing proliferative lesions, are non-enveloped viruses with a double-stranded DNA genome of 7.8 to 8 kilobases. Exophytic genital wart(EGW), one of the most common sex-transmitted diseases(STD) worldwide, is caused mainly by human papillomaviruses type 6 and 11(HPV-6, 11) infection of anogenital areas. EGW is very popular in the world and is the second prevalent STD in China. Patients with EGW suffer greatly in both physiology and psychology, paying considerable medical expenses for the therapy. Many treatments for EGW are currently in clinical use including cryotherapy, surgical excision and drug therapy etc., but recurrence was observed and non of them is proved to be highly efficient for anyone in any clinical cases. Therefore, it is of significance treating and preventing EGW. Seeking more effective, specific and preventative means is future research direction for control of EGW. Development of vaccines against EGW has been a pivot for the last five years.Because virions from natural lesions are scarce and it is now impossible to propagate HPV by animal infection or to obtain abundant virions by in vitro culture systems, the availability of virus particles for vaccination studies is limited. This problem has been overcome by genetic engineering production of HPV subunit vaccine. HPV capsid is composed of the major capsid protein L1 and minor capsid protein L2 in a ratio of 10:1. Previous study have demonstrated that L1 or L1 plus L2 can assemble into virus-like particle(VLP) when expressed in eukaryotic cells. VLPs are structurally and antigenically similar to naturally occurring virions but without virus DNA. VLP preserves conformational epitopes from authentic virions, is highly immunogenitic and can induce preventative neutralizing antibodies in animals. The research in past five years or so testified VLP as the most promising candidate vaccine against HPV. L1 can not assemble into VLP when expressed in E. coli. only can the expressed product do so after complicated process of denaturing and renaturing with the efficiency very low. We used baculovirus-insect cells expression system to express HPV-6 L1 and L2 gene that were cloned from Chinese patients with condyloma acuminatum, questing the feasibility of developing VLP based preventative vaccine against EGW. The following are results we obtained:1. Cloning and sequence analysis of HPV-6 L1 and L2 geneHPV DNA was extracted from biopsies of Chinese patients with condyloma acuminatum, the type was determined using methods of type-specific PCR and subsequent restriction analysis. Using overlap PCR design, major capsid protein (L1) and minor capsid protein (L2) genes were separately amplified from HPV-6 positive samples, and were further assembled...