| Human papillomaviruses(HPVs) are double-stranded DNA tumor viruses with specific tropism for keratinocytes that induce hyperproliferative lesions in cutaneous and mucosal epithelia. They spread through direct and indirect contact. Condyloma Acuminatum(CA) is the most common sexually transmitted disease by infected with HPVs and its infection rate is very high. Infection with a human papillomaviruses, particularly the subset of "high-risk" viral types, can account for the subsequent development of carcinoma and cervical cancer. Expeciously HPV 16 can be detected nearly 50% among cervical cancer's tissues. Because there is no effective precaution and remedy for the diseases by infected with HPVs and cervical cancer, hardly acquired amount of papillomavirus particle in vitro now as well as there are no animal model which natural infected by HPVs, the work for studying HPV vaccines is limited.To establish the animal model for studying HPVs and evaluating its vaccines, we extract DNA from fresh cervial cancer tissue of Jiangsu district and acquired gene E6E7 and LI by PCR, and established E6E7 transgenic mice models and LI expression tumor models. We constructed recombinant plasmid pcDNA-Ll, pcDNA-E7 and tested their protective immunity. We summarize them from four sides below.Part one: Clone and Sequence Analysis of HPV16 Type E6E7 and LI GeneAfter extracting genome DNA from fresh cervical cancer tissue of Jiangsu district, we designed two specific primers according to the genome DNA sequence of HPV16 type standard strain have been reported, including initial and end codes, and acquired gene E6E7 and LI by PCR . The PCR products were cloned into pGEM-Teasy vector routinely, and the positive recombinants were identified by blue-white screening and endonuc lease digestion. Then the E6E7 gene and L1 gene we got were sequenced and analyzed. We constructed recombinant plasmids pGEM-T-E6/7 and pGEM-T-L1, the results show that these genes are highly homogeneous with German standard strand. The sequence of E6 is as same as German standard strand, there is only one mutation in 199th bp(T→C) of E7 and the mutation doesn't cause any change of amino acid, there are seven mutations of LI and four mutations change the amino acids as below: 6240th (bp C→G aa H→D ), 6432th (bp A→G aa T→A), 6903th -6905th (insertion, bp CAT, aa S), 6954th -6956th(deletion, bp GAT, aa D).The successful clone of the HPV16 E6E7 gene and L1 gene will enrich the molecular epidemiological data about HPV16 of our country and are the fundamental study of HPV genetically engineered vaccine with independent intellectual property. These works also lay the foundation to establish the HPV 16 E6E7 transgenic mice tumor model and HPV 16 L1 Antigen Expressing Tumor Model.Part two: Construction of human papillomavirus 16 E6/7 transgenicmiceAfter extracting genome DNA from fresh cervial cancer tissue, we acquired promoter gene of Human Involucrin(hlNV) by PCR. Using techniques of gene recombination, delete P_cmv promoter from pCEP4 vector and replace P_INV promoter, constructed vector contained P_inv promoter pCEP4/P_INV (Pcmv ). Cutting the E6/7 gene from pGEM-T-E6/7 and inserting its into MCS of pCEP4/P_INV(P_cmv), the recombinant eukaryotic expression plasmid pCEP4/P_hINV/ E6/7 ( -Pcmv ) was acquired. After that, we verified the tissue speciality of pCEP4/P_hINV/ E6/7( -Pcmv ) with transfection to severalkinds of cells. To establish the P_hINV/E6/7 transgenic mice, we microinjected the Pinvpromoter-HPV16E6/7-SV40 PolyA DNA fragment into male nuclei. In this research, total 129 mice were born. By PCR, 5 transgenic mice were detected positive forP_hINV/E6/7( 44 # , 47 # . 99 # , 106 # , 108 # ). To confirm the positive transgenic mice, southern blot was used to detect these positive transgenic mice. The result showed that 4 transgenic mice were positive for P_hINV/E6/7 ( 44 #, 47 #, 99#, 106#). After propagating, the F1 positive rate is 50.3%(19/36), the F2 positive rate is 53%(62/l 17) by PCR detection, which suggested that this was conformed to the laws of...
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