| Background: Colorectal carcinoma is one of major tumors which severely damage the health of mankind, and its major therapy is through surgical intervention aided by radiotherapeutics and chemotherapeutics. But recrudescence of tumor is easy to take place, especially the metastasis of liver. It is reported that the survival rate of colorectal carcinoma patients with liver metastasis is lower than 30% five years after the operation. Along with the rapid progress of molecular biology and genetic engineering, gene therapy is becoming one of effective approach to control malignant tumor. Among all the approaches of gene therapy, suicide gene therapy which is interesting more and more researchers has become the focus of study in recent years. But the effect of suicide gene therapy is not satisfactory because of low efficiency of traditional gene vector such as retrovirus, liposome and the tolerance of single gene. As the newly emerging vector, recombinant adenovirus is being given more attention and has become the major vector of gene therapy because of its high rate of transfection and expression.Objective: The traditional method of adenovirus construction through homologous recombination in cell is not only complicated and difficult but also less successful. A little bit more successful (20%) and easy way adopted by He TC et al is to recombinate homologously in bacteria. This study is to construct the recombinant adenovirus of double suicide gene driven by CMV promoter which is applied in the therapy of colorectal carcinoma. Methods: This study has designed a two-step CaC12 transformation method based on homologous recombination in bacteria to ensure a higher rate of successs. CD and TK gene were amplified by PCR and then were cloned into shuttle vector pAdtrackCMV. The Pme I-linearized resultant plasmidpAdtrackCMV-CDTK was transformed into competent AdEasier-1 cells prepared by method of CaC12. The DNA of identified recombinant plasmid was digested with PacI and tranfected into 293 cells to package adenovirus. PCR technigue was used to detect target gene. After liter of the adenovirus, there were used to infect Lovo cells in vitro for killing test. Results: The results showed that CD gene (amplified from the template of E. coli JM109 by PCR) and TK gene (amplified from the template of pREP8-TK plasmid by PCR) were identical with the sequences reported. Chemical transfortiom of linearized transfer plasmid into AdEasier-1 cells resulted in very high success rate(20/20) of recombinant adenovirus plasmid. PCR test indicated that the recombinant adenovirus contained CD and TK fusion gene. DNA sequencing analysis indicate that the size of CD/TK refusion gene is 2439bp, without any introns and coding 813 amino acids. 50% pecent of Lovo cells express green flurosecent protein(GFP) 24 hours after transfection of recombinant adenovirus. When treated with 5-FC and GCV, all the GFP expression cells were eradicated and some portion of non-GFP expressing cells were killed four days later which was called bystander effect. At the same time the group without 5-FC and GCV showed GFP expressing, no cell killing. Conclusion: These experiments demonstrate that the two-step CaC12 transformation method is more convenient and efficient in construction of recombinant adenovirus and recombinant adenovirus containing double suicide gene driven by CMV promoter can effectively mediate target gene expression in transfected cell thus paves a sound foundation for further study.
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