Cloning And Expression Of SV40 T Gene In Escherichia Coli And Application Of Its Polyclonal Antibody On Transgenic Mice

Simian virus 40 (SV40), a small DNA virus, was discovered and isolated from the monkey kidney in 1960s. SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro. And to be associated with certain types of human cancer, including mesotheliomas and brain tumors, etc. SV40 can induced tumors in transgenic mice. In recent years, a great deal of oncology research has focused on the relationship between simian virus 40(SV40) and human tumors. In order to study the association of SV40 with human cancers, we established SV40 transgenic mice in our laboratory. Pancreas cancer was found in these models of the transgenic mice. Moreover, these transgenic mice provide the tools of studing human tumors for the researchers.In this essay , the fragment of SV40 T gene was amplified from total RNA of COS-1 cells by RT-PCR method, and then cloned into pGEM-T easy vector. After DNA sequencing, it was subcloned into the downstream of GST gene in expression plasmid pGEX-6p-l. The recombinant vector was transformed into E. coli BL2i , GST fusion protein was expressed at the induction of IPTG The size of this fusion protein was 53ku which confirmed by SDS-PAGE and Western blotting. The New Zealand rabbit were immunized with the fusion protein to prepare polyclonal anti-serum which was checked by indirect immunofluorescent assay(IFA). The result showed that the anti-serum of the rabbit can response to the SV40 T antigen of COS-1 cells. This study suggested that the expressed products of SV40 T gene in Escherichia coli with pGEX-6p-l vector hadspecific antigenicity of SV40 TTo detect the expression of SV40 large tumor antigen in transgemc mice, the anti-serum of rabbits was used as the first antibody (polyclonal antibody IgG) in the method of immunohistochemistry. SV40 large tumor antigen was expressed only in the Pancreas cancer cells, and most of them were lied in nucleus. The result proved that SV40T antigen has cell transformation properties in transgenic mice in vivo. This result was conformed to some references. Other tissues and organs were negative for SV40 large tumor antigen, such as liver, brain, heart, spleen, kidney, lungs. So. the anti-serum of rabbits (polyclonal antibody IgG) can be used for detection of SV40 transgenic mice in immunological field. Therefore, the transgenic mice can be checked not only at DNA level, but also at protein level.