Expression And Purification Of Human Papillomavirus Type 58 Late Protein L1 In E.coli

The human papilloma virus is can cause the human skin, themucous membrane papilloma or a wart kind of tumor relatedvirus. At present, HPV has separated more than 100 types.Among them, infection genital tract HPV acts according to itspathogenicity to be possible to divide into the highly risky typesand the low risky types. The low risky types mainly includes 1, 2,6, 11 and so on, mainly causes the benign lump or the wart, likeflat wart and incisive condyloma;The high risky types mainlyincludes 16, 18 and so on, mainly causes the skin mucousmembrane malignant tumor, like cervical cancer and so on.The recent years literature reported and the epidemiologyresearch indicated, has the high carcinogenicity risky besidesHPV16 and 18, but also some 31, 33, 45 and 58. Internationalstudies have consistently showed a high prevalence of HPVinfection among women with invasive cervical cancer and thatHPV DNA can be detected in approximately 90% of al1 cervicalcancers. Recently, strong evidence from epidemiologicalsurveysand molecular biological studies has implicated thathuman papiL1omavirus type 58(HPV58) infection is closelyrelated with cervical cancer. Particularly, in SouthernChina, thepositive rate of HPV58 was the higher in biopsies of cervicalcancer. The positive rate of HPV58 increased gradual1y with themalignant extent of the lesions assayed, which is the same as thatof HPV16. HPV58 has been shown to be one of the mostimportant highly-oncogenic, risky HPV types in China.At present, cervical cancer oneself becomes endangers oneof woman health main malignant tumors, the disease incidencerate has the tendency which advances year by year, but becauseHPV still could not pass through the tissue culture until now tocarry on the multiplication, also the viral nucleic acid includedthe latent carcinogenicity, therefore was unable to prepare thenatural vaccine to prevent. On clinical high risk type of HPVinfects the easy canceration, in front of the cancer thepathological change to use the medicine treatment result to bebad, spends high;To the middle or later period cervical cancer ofchemotherapy, the surgery treat is not ideal. Therefore, studiesand develops the HPV high risky types genetic engineeringvaccine watershed.HPV prophylactic vaccine research oneself receives thedomestic and foreign medical arenas universal attention.Chooses the ideal goal gene to develop the genetic engineeringvaccine to be extremely important. The HPV genetic engineeringvaccine goal gene choice mainly concentrates in early gene E6,E7 and later period gene L1, in the L2 gene.The therapy vaccineoften applies E6, the E7 early gene, but the preventive vaccineoften applies L1, the L2 gene. The HPV later period gene areacodes two structure proteins: Main capsid protein L1, thestructure is conservative, is the main kind of specificity antigen;minor capsid protein L2, is the specificity antigen. The L1 Majorcapsid protein has the conservative nature as well as with thenatural viral pellet similar antigen epi-position, therefore can beinduced in the protection and the immune body, may preventHPV the infection. Therefore, the HPV prophylactic vaccineapplied research mainly concentrates at present on HPV outercovering protein L1.At present, domestic and foreign to HPV16, 18 has done themassive work, but to the HPV58 research, specially the HPV58structural gene constructs, the expression and the geneticengineering vaccine research are extremely few. Based onHPV58 in the our country woman cervical cancer specialposition, as well as the clinical treatment faces all sorts ofdifficulties, the research develops the HPV58 preventive vaccine,then finally develops the HPV high risky type the union vaccine,to prevented HPV infects and the malignant tumor which causesby it has the significant practical significance.This article uses this laboratory preservation the HPV58pathology tissue to extract genomic DNA, then amplified by thePCR the full L1 gene fragment, inserting the HPV58 L1 geneinto pGEM-T and identified with restriction analysis, thensubclones to expressing plasmid pRSET-B. The recombinantexpression plasmid pRSET-B-L1 was successfully constructedand then transformed to BL21 (DE3), HPV58 L1 major capsidprotein was efficiently expressed in E.coli by IPTG induction.SDS-PAGE analysis result showed special band of theinduced product was observed at the position of Mr 58 KD, andthe protein was expressed mainly in the form of inclusion bodies,Western blot confirmed expressed the protein the specificity.This research design selects the HPV58Ll gene to constructthe recombinant expression plasmid, expresses the HPV58L1Major capsid protein, purifying L1 major capsid protein bychromatographically. This study provides a good basis foridentification of the biological function and preparation ofHPV58L1 specific antibody and VLPs assembly and thepreparation corresponding immune body from now on usingthe L1 protein, then developed the HPV58 genetic engineeringvaccine and the HPV multi-valence vaccine has laid the goodfoundation and the condition.