Construction Of Recombinant Adenovirus Containing Hepatocellular Carcinoma Specific Co-expression Of SEA And CD80

Primary hepatocellular carcinoma (HCC) is one of common malignant tumors in China, and nearly 45% of emerging patients with hepatocellular carcinoma are in China mainland every year, and there is an increasing trend in recent years. Statistics show that the annual mortality of HCC in China is about 20.40/10 million, and account for in the first and second cancer mortality in urban and rural areas, respectively. The surgerical methods couldn't completely remove all tumors now, while radiotherapy, chemotherapy and other traditional methods are not conducive to a thorough treatment of cancer. With the development of biotechnology and gene therapy, the biological treatment of HCC become an important means. By stimulating anti-tumor immune reaction, immunotherapy shows a very bright future, and become "green channel". However, the tissue-specific targeting must be resolved before the clinical application of gene therapy, which could reduce the side effects. In addition, because of the weak immunogenicity of HCC cells, low expression and lack of MHC molecules and costimulatory molecules, current immunotherapy often could not induce a more effective immune response, and cancer cells could escape immune system identification and attack. Superantigen could activate a large number of T cells by directly binding the MHC-II molecular and TCR of T cells as a complete protein molecule without antigen processing of presenting cells. Staphylococcal enterotoxin A(SEA) is superantigen, and it has been reported in treatment of human tumors. CD80, an important stimulatory molecule, could interact with CD28 molecular on T cell surface, and give the second signal to activate T cells.In this study, we constructed the recombinant adenovirus containing hepatocellular carcinoma specific co-expression of SEA and CD80. AFP promoter/enhancer was used as a cis-activating element to regulate the coexpression of CD80 and the transmembrane SEA genes, and the Ad5 adenovirus system was introduced as a carrier, which laid the foundation for further experiments for gene therapy of hepatocellular carcinoma.1. Construction of adenovirus shuttle plasmid containing human AFP promoter and enhancer.Objective: To construct adenovirus shuttle vector containing alpha-fetoprotein (AFP) promoter/enhancer sequences as cis-regulatory element. Methods: Design the specific primers of AFP promoter and enhancer, using human liver genomic DNA as template to amplify AFP promoter and enhancer fragments. These fragments were subcloned into revised shuttle vector pShuttle2 by ligation, transformation, and other steps to construct adenovirus shuttle vector pShuttle-AFP. Results: The restriction enzyme digestion and gene sequencing showed AFP promoter/enhancer sequences were correct and identical with the corresponding sequences published in Genebank. Conclusion: We successfully constructed the adenovirus shuttle plasmid pShuttle-AFP carrying AFP promoter/enhancer sequences, which could be used for further treatment of liver-specific gene therapy.2. Construction of adenovirus shuttle plasmid containing co-expression of transmembrane SEA/CD80 regulated by AFP enhancer/promoter.Objective: To construct adenovirus shuttle vector containing co-expression of SEA and CD80 genes regulated by AFP enhancer and promoter. Methods: Designing the specific primers of SEA and CD80 genes, respectively, and using pMD18-tmSEA vector and human liver cDNA library as templates to amplify transmembrane SEA (tmSEA) and CD80 genes. The tmSEA fragment was subcloned into the pIRES2-EGFP vector to construct pIRES2-EGFP-SEA, and immunofluorescence was used to determine the subcellular localization of SEA. The CD80 fragment was ligated into pMD18-T vector, to construct plasmid pMD18-T-CD80. The plasmids were analyzed by restriction endonuclease and sequencing. CD80 fragments from pMD18-T-CD80 and SEA-IRES fragment from pIRES2-EGFP-SEA were inserted into pShuttle-AFP vector to construct adenovirus shuttle plasmids pShuttle-AFP-SEA-CD80. Results: The cloned SEA and CD80 sequences were identical with those published in Genebank. All of the restriction digestion and PCR analysis showed the expected results. At the same time, immunofluorescence showed the plasma membrane localization of trans-membrane SEA in HepG2 cells. Conclusion: We successfully constructed adenovirus shuttle vector containing co-expression of SEA and CD80 genes regulated by AFP enhancer and promoter..3. Construction of recombinant adenovirus containing hepatocellular carcinoma specific coexpression of SEA and CD80Objective: To construct recombinant adenovirus containing hepatocellular carcinoma specific coexpression of SEA and CD80. Methods: AdEasyTM adenovirus system was used in this experiment. The shuttle plamsid pShuttle-AFP-SEA-CD80 linearized by Pme I were recombinated with adenovirus backbone pAdEasy-1 in E. coli BJ5183 to construct pAd-AFP-SEA-CD80 recombinant adenovirus plasmid. Linearized pAd-AFP-SEA-CD80 were transfected into AD293 packaging cells to generate recombinant adenovirus containing hepatocellular carcinoma specific coexpression of SEA and CD80, and amplification. The specific co-expression of transmembrane SEA and CD80 in HepG2 cells infected with adenovirus were detected by Western blot. Results: The constructed pAd-AFP-SEA-CD80 recombinant adenovirus vector could released a 4.5kDa or 3.5kDa expected fragments by restriction digestion with Pac I. PCR analysis showed the expected results. The specific products of tmSEA and CD80 could be detected in HepG2 cells but not 293T cells infected with adenovirus. Conclusion: We successfully constructed recombinant adenovirus containing hepatocellular carcinoma specific coexpression of SEA and CD80, and could specifically express the targeting genes of tmSEA and CD80 in HepG2 cells which produce AFP. The constructed adenovirus provided the materials of immune gene therapy specific for hepatoma.In this study, human AFP promoter/enhancer sequences were cloned from the human liver genomic DNA libary, and successfully constructed adenovirus shuttle plasmid pShuttle-AFP. The transmembrane SEA and CD80 genes were successfully cloned into the pShuttle-AFP, and constructed pShuttle-AFP-SEA-CD80 shuttle vector. Recombinant adenovirus vector pAd-AFP-SEA-CD80 using AdEasyTM adenovirus system constructed by recombinating pShuttle-AFP-SEA-CD80 and pAdEasy-1. Recombinant adenovirus was packaged and amplified in AD293 cells, which could specifically coexpress transmembrane SEA and CD80 in HepG2 cells expressing AFP. In this study, recombinant adenovirus containing hepatocellular carcinoma specific co-expression of SEA and CD80 were constructed, which provided the basis for targeted gene therapy of hepatocellular carcinoma.