HPV58 Infection In Cervix Exfoliated Cells And Construction Of Recombinant Virus With HPV 58 E6E7

Objectives 1、 To understand Human papillomavirus(HPV)infection in exfoliated cervical cells,and explore the relationship between high risk-hpv(hr-hpv)infection and cervical cytopathological features.2、The recombinant virus carrying the HPV58 m E6E7 fusion gene was constructed using HPV58 m E6E7 as target and Towne cytomegalovirus bacterial artificial chromosome(SW102-T-BAC)as vector,and the transformation activity of HPV58 m E6E7 was identified,which lay the foundation for the research of therapeutic vaccine for HPV58 infection.Methods 1、The cervical exocellular cells were collected for physical examination of women.HPV infection was detected by PCR using HPV L1 MY09/11 primers and every sample DNA as template;HPV type was detected by PCR using HPV E6 specific primers;the relationship between HPV infection and clinicopathological features was analyzed by statistical analysis.2、The PCR amplification of Galk cassette and HPV58 E6E7 fusion gene cassette with 50bp(Towne)open reading frame(ORF)75 homologous arms.The desired fragments were obtained by gel cutting,recovery and purification using QIAquick Gel Extraction Kit.Galk cassettes were electroporated to SW102-T-BAC electrocompetent cells,and the clones of SW102-T-ORF75-Galk-BAC were obtained by homologous recombination and screening in Galk and chloramphenicol resistance medium;SW102-T-ORF75-GalkBAC electrocompetent cells were prepared;the m E6E7 and w E6E7 cassettes were electroporated to SW102-T-ORF75-Galk-BAC electrocompetent cells,the clones of SW102-T-ORF75-HPV58-m E6E7-BAC and SW102-T-ORF75-HPV58-w E6E7-BAC were obtained by homologous recombination,selection from replaced Gal K and chloramphenicol medium,respectively.The plasmids of SW102-T-ORF75-m E6E7-BAC and SW102-T-ORF75-w E6E7-BAC were extracted,and transfected into ARPE-19 cells.The expression of m E6E7 and w E6E7 in transfected cells were verified by reverse transcription PCR and sequencing.The effect of T-ORF75-m E6E7 and T-ORF75-w E6E7 on ARPE-19 cells were observed.The transformation activity of m E6E7 and w E6E7 were analyzed by soft agar cloning.Results 1、573 samples were HPV positive by PCR amplification,the total infection rate of HPV was 56.85%.The infection rate of HPV16 type was 7.33%,the infection rate of HPV18 type was 2.62%,and the infection rate of HPV58 type was 13.61%.HPV positive rate of the 41-50 years group was the highest about 63.66% in all age groups.Statistical analysis of HPV infection in all age groups showed that the difference was statistically significant(P<0.05).According to the cytological staging results,the HPV positive rates were 100%,80.15%,51.69% and 51.55% in HSIL group,LSIL group,ASC group and NILM group,respectively,HPV positive rate is decreased with decreases of lesions degree,the difference was statistically significant(P<0.05).The HPV58 positive rate was higher about 12.50% in HSIL group.2 、 Clones of SW102-Towne-ORF75-m E6E7-BAC and SW102-Towne-ORF75-w E6E7-BAC were obtained.Reverse transcription PCR and sequencing analysis verified that m E6E7 and w E6E7 were expressed correctly in transfected cells.The cells transfected with SW102-t-ORF75-w E6E7-BAC lost contact inhibition and showed overlapping growth.The morphology changed from the original spindle deformation to a round shape with swelling and increased cytoplasmic granules.Moreover,clones could be formed in soft AGAR.However,SW102-Towne-ORF75-m E6E7-BAC and SW102-Towne-BAC and non-transfected cells did not show the characteristics of these cells.Conclusions 1、The total HPV positive rate is 56.85% in the collected samples,among them HPV58 positive rate is about 13.61%,which is higher than that of other HPV types.HPV positive rate was 63.66% in 41-50 year group,which is higher than that of other year groups,and the overall HPV infection rate increased with the aggravation of cervical lesions.Therefore,it is necessary to study HPV therapeutic vaccine.2 、 The recombinant virus of T-ORF75-HPV58-m E6E7 and T-ORF75-HPV58-w E6E7 were obtained,and the transformation activity of recombinant virus of T-ORF75-m E6E7 has been eliminated,which provided a foundation for the research of HPV58 therapeutic vaccine.Figure [14];Table [5];Reference [86]...