Molecular Epidemiology Investigation Of HPV And Establishment Of The Double Antibody Sandwich ELISA Detection Method In Parts Of Northern Xinjiang

Cervical cancer is one of the most common female malignant tumors, the persistent infection of high-risk HPV(HR-HPV) is closely related to the occurrence of cervical cancer. Currently the prevalence of cervical cancer gradually increased, and showed a trend of strengthening, which not only affect seriously the quality of life, but also formed a serious threat to the health of women. So it is important to investigate the relationship between HPV infection and the cervical lesions and to analyze the prevalence and genetic variability of epidemic HPV genotype. In order to promote the application of the HPV vaccine and study a new generation of vaccines, and also establish a new testing method to detect the infection of HPV. Xinjiang, located in Northwest China. There has a relatively higher incidences of cervical cancer, especially in southern xinjiang minority nationality areas. However, at present, the research on epidemiological data and vaccine of HPV in northern region of Xinjiang remains unclear.In this study, the prevalence of HPV in parts of northern region of Xinjiang, the distribution of HPV genotypes in cervical lesions in the sample and the relationship between multiple infection and cervical lesions were studied. Based on this research, vaccines suitable for this region and new detection methods for HPV will be studied by analyzing the genetic variation. The methods and results are as follows: 1. The prevalence of HPV in parts of northern XinjiangThe persistent infection of HPV is the major factor of cervical cancer. In order to investigate the prevalence of HPV, the distribution of HPV genotypes and the multiple infections in cervical lesions a total of 7,298 samples from women who were enrolled for the study using the Hybrid Capture 2(HC2) test in the First Affiliated Hospital, School of Medicine, Shihezi University were collected and analyzed. 2046 cases of cervical cytology specimens in 2014 were collected. People with cervical lesions were detected by PCR. The results showed that:(1) the incidence rate of HPV was still at a higher level, especially focus on the population who is the age in < 25 and ≥ 55;(2) the infection prevalence of HR-HPV increased with the severity of pathological diagnostic grades. The infection prevalence of LR-HPV types, however, did not change notably between different grades of pathological diagnosis;(3) there are at least 29 kinds of HPV genotypes in northern Xinjiang, including 17 kinds of high-risk types, 12 kinds of low-risk types, and the higher incidence of HR-HPV genotypes were HPV 16, 53, 52, 58, 35;(4) among the multiple HPV infections, double infections, especially combined with HPV 16 is the most common. The triple, quadruple, pentagonal and hexagonal infections have also appeared, but the multiple HPV infection was not necessarily correlated with the severity of cervical lesions. 2. Cloning and sequencing of L1 gene from the epidemic HPVIn order to study the variation of L1 gene from the high risk HPV in northern Xinjiang the L1 primers of HPV 16, 52, 53, 58 as well as a higher incidence and stronger carcinogenic HPV 18 type were designed. In order to amplify the full length sequence of L1, the full-length sequence of L1 gene were obtained after the sequence Assembly. Through comparing the sequence of the existing vaccine strains and the United States strains, the mutation was found and some mutations even lead to amino acid change. Take HPV 16 for example, the differences were found between the Xinjiang strains and vaccine strains by the molecular bioinformatics analysis. These results will afford a theoretical basis for introducing vaccines and developing the multivalent vaccines for HPV of North of Xinjiang. The study also reported there was a good conservative screened polypeptide fragment when compared the past study, and lay the foundation for the preparation of monoclonal antibodies in the next chapter. 3. Detection of monoclonal antibodies of HPV 16 and establishment of the double antibody sandwich ELISA detection method for HPVIn this study, the polypeptide fragment was choosed in the second experiment, and then mithridatized mice after coupled with KLH, 15 monoclonal antibody-containing hybridoma cell culture supernatant were obtained. Only two positive clones were got by western blot testing. With the help of the purified monoclonal antibody and the HRP-labeled monoclonal antibody, the double sandwich ELISA detection method was developed to detecte HPVL1 in northern Xinjiang. The linear detection range was 1.69 μg/mL purified polyclonal antibody, and HRP-labeled monoclonal antibody. The sample and HRP-conjugated antibody reaction time were 45 min and 40 min, respectively. And the chromogenic time was 15 min. The agreement rate from sixty-six clinical cervical cell samples detected by the ELISA and TS-PCR was 81.8%. The results revealed that the double sandwich ELISA had good specificity and repeatability, which could lay the foundation for the establishment of a quick and easy HPV detection method.