Constructing Prokaryotic Expression Plasmid And Sequence Analysis Of L1 Major Capsid Protein Gene Of Human Papillomavirus In Relapse Condyloma Acuminata Patients

Objectives To construct prokaryotic expression plasmid of late gene L1 in relapse condyloma acuminata patients and establish the foundation for gene expression of L1 by providing useful dates in studying the biology characteristic of HPV L1 protein . In addition, we want to analyze the structural specificity of L1 major capsid protein gene of human papillomavirus in relapse condyloma acuminata patients by sequence analysis and comparing with the original sequence.Methods DNA templates were purified from genital warts lesions of four patients. A conservative 233bp long sequence between E6 and E7 was amplified by PCR and digested by restriction enzymes Rsa 1 in order to typing . Then we picked DNA sample from the patient infected with HPV6 as templates to amplify full coding L1 sequences by PCR. Having been digested by restriction enzymes Bam HI and Hand Ⅲ, HPV6 fragment and linearized expression vector pQE40 were ligated to construct recombinant prokaryotic expression vector which it were transformed into coli DH a . then. The recombinant vector was identified by Bam H I and Hand III digestion. The positive vector was sequenced to get late gene L1 sequence. Results There were three cases infected with HPV 6 and one case infected with HPV11. It showed that HPV 6/11 are the main causes of genital warts. HPV L1 gene was successfully cloned. Compared with the prototype accepted by GenBank, mutations were found in 7 bases of L1 gene which resulted in 3 changes of corresponding coding amino acids. The 6062 base of L1 gene changed from A to G, which leading to the change from Threonine to Alanine. The 6684 base of L1 gene changed from A to T which leading to the change from Glutamic acid to Valine. The 6945 base of L1 gene changed from T to C which leading to the change from Isoleucine to Threonine. The rest four were all same-sense mutations: A →T at nt 6598, A→G at nt 7081, G→A at nt 7099 and G→A at nt 7243. Conclusions The construction of prokaryotic expression plasmid of HPV6b L1 was achieved. Variations take place in the nucleotide sequence of HPV6 L1 gene from the relapse condyloma acuminate patients especially the lower sequence. The mostfrequent substitutions from HPV6b LI ORF sequence of relapse condyloma acuminata appeared to accumulate within one discrete region: R3 (nt 7070-7230) and SR4, which reported by Wanderly and Lin Shaohong respectively. It suggested that this region is easy to appear substitutions in spite of the region of Rl and R2 is just as long as 1 OObp,...