| Study on Human Papillomavirus Type 16 E7 DNA VaccinesHuman papillomavirus type 16 is a dominating pathogen of cervical cancer. Being oncogenic virus, it has also been established a close association with several types of malignant human tumours. HPV16 E7 is a kind of important suffering cancerous gene and at the same time it encodes a transformed nonstructural virus protein. This oncoprotein is involved in malignant transformation of cervical carcinoma cells and its presence is required for the maintenance of the malignant phenotype of the cells; and the expression level of the E7 protein correlates with its transforming potential. Therefore, if HPV16 E7 was being used for construction of the papilloma virus vaccines, it will lead to a new strategy to prepare to prophylactic and therapeutic vaccine against cervical carcinoma. HPV16 E7 protein has several motifs stimulating specific B cell, CD8+ T cell and CD4+ CTL immunity. With high affinity to MHC molecule, the utility of E7 as the tumour specific rejective antigen can stimulate significantly stronger immunological reactions against HPV16 induced disease. The research contains: cloning and mutations of HPV16 E7 gene isolated from some Chinese Uygur patients of cervical carcinoma in South Xinjiang, expression of HPV16 E7 fusion protein, preparation and identification of HPV16 E7 antiserum, and immunological evaluation on mice of wild and different mutated HPV16 E7 gene vaccines.At first, according to standard HPV16 E7 gene sequence published by Genbank, a pair of special primer was designed for PCR of HPV16 E7 DNA isolated from the cervical carcinoma tissues of Xinjiang Uygur women. The HPV16 E7 gene was cloned into pMD18-T cloning vector, positive clones were analyzed withrestriction enzyme digestions and further identified with sequence analysis. Thesequencing results show that the HPV16-E7 from Xinjiang Uygur women was identical with the published sequence derived from the Germany strain (GI: 9627100). Then, a constructed prokaryotic expression plasmid of pGEX-2T-E7 presented by Dr. Werner, was transformed into E.coli BL21(DE3 stain) and induced by IPTG to express GST-E7 fusion protein. The expressed GST-E7 fusion protein was purified from bacterial lysates by affinity chromatography using glutathione-agarose resin. This purified and quantified fusion protein which is showed one band by SDS-PAGE, be used to immunize Zelanian rabbits for prepare antiserum on several times. By ELISA analysis and Western blot detection, the liter of this rabbit's polyclonal antibody is 1:60000, and this serum can be identified by HPV16E7 proteins from prokaryotic and eukaryotic expression system.Since the HPV16 E7 protein is capable of transforming somatic cells, utilization of wild HPV16 E7 DNA for vaccination is not safe. The E7 gene can be changed by deletions, insertions or mutations, hence, the mutated, non-transforming HPV16 E6/E7 DNA can be used as a vaccine. Three pairs of primer with the desired mutations were adopted to generate the mutant clones for HPV16 E7 gene, The mutations are introduced from original Ts to Gs at 70, 172 and 271 nucleotides of HPV16 E7, that resulting three amino acids changes at the 24, 58, 91 of HPV16 E7 protein sequence. The mutations introduced to HPV16 E7 gene can be used to develop a vaccine for HPV infection and immunotherapeutic approaches in future.For construction of DNA vaccine, wild and different mutated HPV16E7 genes were subcloned into a eukaryotic expression vector of pCDNAS.O. Utilizing these different eukaryotic expression plasmids of pCDNA3-E7(W), pCDNA3-E7(S), pCDNA3-E7(D), pCDNA3.0-E7(T) to immunize NIH female mice in order to evaluate the immune responses of these constructions, the animals were inoculatedintramuscularly by these DNA vaccines, mice sera and spleens were collected to analyzed their specific humoral and cell-mediated immunity by using ELISA assays and RT-PCR screen. The results showed that wild and different mutated HPV16E7 genes vaccines have been identical to antibodies again... |
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