| Cervical cancer is the second most frequently found neoplasia in women worldwide and also is the No.1 killer in some developing countries. Cervical cancer morbidity and mortality have decreased substantially mostly due to early diagnoses and treatment of cervical dysplasia and cervical neoplasm in developed countries. However in developing countries such as China, the morbidity is 6 times of developed countries, because of unsuccessfully organized or opportunistic screening with Pap cytology. HPV is considered the first necessary cause of cervical cancer. About 99.7% of cervical cancers contain viral DNA, which, in 80% of cases, belongs to highly oncogenic types: 16, 18, 31 and 45. And approximately 50% of cervical cancers are associated with HPV16 infection. Thus an effective vaccine against HPV16 can be used to prevent and treat the cervical cancers. Now there are two main strategies to develop cervical cancer vaccines, prophylactic and therapeutic. It was found that recombinant late L1 protein of HPV alone and L1 protein in combination with L2 protein of HPV could resemble into true HPV called virus-like particles (VLPs). The VLPs was proved to be excellent candidates as prophylactic vaccines against HPV. In clinical trials, HPV16 virus-like particle vaccines were well tolerated and could stimulate human body to generat high levels of antibodies especially sIgA against HPV16. The prophylactic vaccine will come into use in the near future. Therapeutic vaccines, on the other hand, are expected to have an impact on advanced lesions and residual illness of cervical cancer, by taking advantage of the fact that early E6 and E7 genes of HPV constitutively expressed in cells of cervical tumors and precancerous lesions. Thus, an efficient approach to develop vaccines against HPV16 associated tumors is to produce HPV E6/E7 based recombinant proteins.It is realized that cellular immunity is more important than humor immunity in anti-tumor response. In immune system, Cytotoxic T lymphocytes (CTLs) are the most powerful tumor killers and only recognize the epitopes associated with MHC I molecules on the surface of antigen presenting cells or target cells.In this project, we selected 8 CTL epitopes from E1, E2, E4, E5, E6, E7, L1 and L2 of HPV16 based on the epitope prediction sofewares in the internet. The epitopes selected are restricted by HLA-A0201, A11, A24 and A2402, most common HLA class I alleles among Han Chinese. An encoding gene of mutiple epitopes of HPV16 are constructed by PCR and fused to the downstreams of TAT, Chapronin 10, TrxA genes to create the TAT-HPV, Chap10-HPV, TrxA-HPV fusion genes respectively. These fusion genes were coloned into pET28a or pET32a vectors and successfully expressed corresponding fusion proteins TAT-HPV, Chap10-HPV and TrxA-HPV.The main contents of this paper are the following:Part one: Design of CTL multiple-epitopes from HPV16We selected the epitopes according to the published articles and bio-software. These epitopes are covering the most common HLA I types of Han Chinese and conservative according to the analysis in database of Genebank. The decision of the combination of the epitopes is based on the parameters in bio-software PROTEIN. Part two: Obtaining the gene of mutiple-epitopes from HPV16HPV0 gene encoded 8 CTL epitopes derived from HPV16 was synthesized by 5 round-PCR. The gene contains restriction enzyme sites, EcoR I and Bgl II in 5' of the gene, BamH I and Hind III in 3' of the gene. The gene is identified with DNA sequencing.Part three: Construction and expression of HPV in the vector pET28a.HPV1 gene was synthesized by adding Nco I in 5' of HPV0 gene and stop codon and Hind III in 3' of it through PCR. HPV1 gene was inserted into the expression vector pET28a and sequenced. The expression plasmid pET28a / HPV was transformed into BL21(DE3) bacteria. The transformed bacteria were cultured, induced by IPTG for 3 hours, harvested and lysed. No visualized target proteins were found in SDS-PAGE.Part four: Construction and express...
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