Genomic Sequence Analysis Of A Human Papinomavius Type 16 Isolated From Uygur Cervical Cancer Patient In Xinjiang, China And HPV16 Particles Are Assembled In 293T Cells

Human papillomavirus(HPV) is a common small DNA tumor virus,whose genome size is about 8kb. It specifically infects squamous epithelial cells and causes benign or malignant epithelial lesions such as genital warts and cervical cancer.High risk HPV is detected in specimens of more than 90% of cervical cancerHigh risk human papillomaviruses Numerous variants of HPV16 have been identified in different geographic locations and ethnic groups,which are different in pathogenecity.There is a very high incidence of CC in southern Xinjiang,High risk HPV ,such as HPV16 and HPV18,are major causes of cervical cancer (CC),and HPV16 was found most frequently in CC patients.This dissertation predominantly included genomic Sequence Analysis of a Human Papinomavius Type 16 Isolated from Uygur Cervical Cancer Patient in Xinjiang, China and HPV16 Particles are Assembled in 293T Cells .The aim was to investigate the relationship of Xinjiang HPV16 mutation with high frequency of cervical cancer in this area, so as to lay a good foundation of development of Xinjiang cervical cancer vaccine and research on mechanism of carcinogenesis, respectively.This dissertation included five parts:(1) Cloning and Sequencing Analysis Human Papinomavius Type 16 Isolated from Uygur Cervical Cancer Patient in Xinjiang;(2) Polymorphism of HPV16 type E1 genes from cervical carcinoma biopsies in Xinjiang Uygur women and cloning as well as expression of core fragment of E1 gene; (3) Construction of codon-optimized HPV16 capsid genes in the eukaryotic co-expression vector and transfection of cells;(4) Research of the Expression Level of XinJiang Strain HPV16 Gene L1 in Eukaryotic Cells; (5) HPV16 Particles are assembled in 293T cells 1.Cloning and Sequencing Analysis Human Papinomavius Type 16 Isolated from Uygur Cervical Cancer Patient in XinjiangThe primers were designed to clone Xinjiang HPV16 ,which were based on HPV16 sequences published in GenBank. Xinjiang HPV16 from Uygur cervical cancer Patient was iso1ated LA-PCR technique. Sequencing result showed that the Xinjiang HPV16 consists of 7963 base pairs (bp) and has a base composition of 2606 adenine (32.7%), 1392 cytosine (17.5%), 1537 guanine (19.3%), 2428 thymine (30.5%), and a GC content of 36.8 %. The nucleotide sequence comparison showed that there was insertion in the Xinjiang HPV16 E1 gene,which is reason that genome of XinjaingHPV16 is longer than other HPV16.2. Polymorphism of HPV16 type E1 genes from cervical carcinoma biopsies in Xinjiang Uygur women and cloning as well as expression of core fragment of E1 geneThe 41 tissues DNA were extracted from cervical carcinoma biopsies. HPV16 E1 genes were amplified by PCR , HPV16 typeE1 genes were sequenced and analyzed. The positive rate of HPV16 E1 was 78%(32/41) . We sequenced DNA found some mutations in comparison with the previously published sequence of prototype HPV16 E1.Some of the mutations changed the triplet codes , subsequently led to changes of amino acids. The mutations of all eleven HPV16 E1 fragments formed eleven patterns at nucleic acid level. Compare to HPV16 prototype , their homology was 96.67%～99.95%. The mutations of all eleven HPV16 E1 fragments formed five patterns at amino acid level , the mutations of 63 repetition in E1 gene among mutations was not reported before.The core fragment of HPV16 E1 was cloned into the multiple cloning site of pMAL-p2x,pGEX-4T-1 vectors, whose size is 1011bp, and successfully expressed in Escherichia coli BL2(DE3). 3. Construction of codon-optimized HPV16 capsid genes in the eukaryotic co-expression vector and transfection of cellsThe amplifed codon-optimized HPV16 capsid genes from 988 plasmid by PCR were cloned into eukaryotic expressing vector pcDNA3.1(+). Thus, eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 capable of expressing HPV L1 gene and L2 gene was constructed. The transcription of capsid genes was observed in vivo and in vitro by hydrodynamics-based transfection and liposome-mediated transfection.Cytopathic effect (CPE) occurred after the transfected with pcDNA3.1-L1-IRES-L2 into 293T cells. Western blot detection showed the recombinant plasmid L1 protein was expressed in 293T cells .4.Research of the Expression Level of XinJiang HPV16 Gene L1 in Eukaryotic CellsStudying on the expression level of Xinjiang HPV16 L1 gene, because there are mang mutations in Xinjiang L1 gene.The eukaryotic expression vector of pcDNA3.1-xj-L1 was constructed and transferred into 293T cells Recombinant plasmid pcDNA3.1-Human-L1 could express in 293T cells by Western blot, but pcDNA3.1-xj-L1 not. It draw that the Xinjiang wild type HPV16 capsid gene had an low expression level in 293T cells. The result makes the helpful exploration in studying the new Xinjiang local vaccine of HPV 16 and the in-depth research of correlated mechanisms.5,HPV16 Particles are assembled in 293T cellsLinear or recircularized XJHPV16 was lonely transfected into 293T cell,or company with codon-optimized HPV16 capsid genes,E2 gene and E5 gene were detected by RT-PCR. Southern blot analysis of linear genomes revealed that genomes were circular regardless of the state of input viral DNA.L1 protein was detected in co-transfected groups, butlonely transfected not.The experiment showed that HPV16 particles are successfully assembled in 293T cells and codon-optimized HPV16 capsid genes were necessary.