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Aim: to construction of eukaryotic expression vector of the human SOD1 (Cu/Zn superoxide dismutase, SOD1) and expression in HeLa cells., Methods: the open reading frame (ORF) of SOD1 was amplified from human peripheral blood by RT-PCR. TA cloning strategy was used to insert the target fragments into pUCm-T vector. The recombinant plasmid was identified and noted as pUCm-T-SOD1. Then, SOD1 was subcloned into pTracer-CMV/Bsd, a eukaryotic expression vector. The plasmid of pTracer-CMV/Bsd-SOD1 was sequenced and was introduced into HeLa cells by Lipofectamine(TM); 2000. The expression of green fluorescence protein (GFP) was observed by the fluorescence microscope. The expression of SOD1 was detected by RT-PCR and Western blot after screening by blasticidin for 4 weeks., Results: the eukaryotic expression plasmid pTracer-CMV/Bsd-SOD1 was successfully constructed. The GFP was observed in transfected cells by the fluorescence microscope. The expression of SOD1 was detected in transfected cells by RT-PCR and Western blot., Conclusion: the recombinant eukaryotic expression vector of pTracer-CMV/Bsd-SOD1 has been constructed successfully which could express GFP and SOD1, respectively, providing a tool for further gene therapy study.

Aim: To evaluate the potential of Daweizi pigs as xenotransplantation dnors from pigs to humans by analyzing the characteristics of SLA-DR genes in Hunan Daweizi pigs., Methods: SLA-DRA and SLA-DRB genes were amplified by RT-PCR, cloned into pUCm-T vectors, sequenced and analyzed through BLAST in NCBI and related software in ExPASY., Results: The SLA-DRA and SLA-DRB genes were 1 177 bp and 909 bp nucleotides in length, which contain opening reading frame (ORF) and encode 252 and 266 amino acids respectively. Comparing the SLA-DRA and SLA-DRB genes with their counterpart sequences of human, the homologies of amino acid sequences were 82% and 73% respectively. The amino acids in SLA DR alpha chain of Daweizi pigs from position 124 to 136, which bind to human CD4, showed only two differences with HLA DRA: a lle-Val change at position 127 and a Ser-Thr change at position 136. The amino acids in SLA DR beta chain of Daweizi pigs from position 134 to 148, which bind to human CD4, were identical with HLA-DRB. Further comparison with SLA sequences published in GenBank indicated that SLA-DRB gene found in Daweizi pigs has polymorphism while the homology of SLA-DRA gene is up to 100%., Conclusion: The cloned SLA-DRA and SLA-DRB in Hunan Daweizi pigs has high polymorphism with HLA-DRA and HLA-DRB in Human, indicates that Daweizi pigs have some advantages as xenotransplantation dnors from pigs to humans.

Aim: To construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected Hela cell line., Methods: The open reading frame (ORF) of TSARG4 was amplified from human testis by RT-PCR. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into pcDNA3.1(+), a eukaryotic expression vector. The recombined plasmid pcDNA3.1(+)/TSARG4 was sequenced and transfected into Hela cell by lipofectamine 2000. After screening culture by G418, stable transfected HeLa cell line was established, and the expression of TSARG4 was identified by RT-PCR and in situ hybridization., Results: The eukaryotic expression plasmid of pcDNA3.1(+)/TSARG4 was successfully constructed and stable transfected HeLa cell line was established. RT-PCR and in situ hybridization result revealed that TSARG4 was expressed successfully in HeLa cells., Conclusion: The recombinant eukaryotic expression vector of pcDNA3.1(+)/TSARG4 has been constructed successfully and stably expressed in HeLa cell line, providing a foundation for further studies on the function of TSARG4 in vitro.

Aim: To express SRG4, a novel mouse spermatogenesis gene in E.coli and purify its fusion protein., Methods: RT-PCR was used to amplify the 586 bp fragment that was located in SRG4 C-end and included a Sad1_UNC like domain. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into PQE-30, a prokaryotic expression vector with 6xHis tag. PQE-30-SRG4 was sequenced and transformed into E.coli M15. The expression of histidine-tagged fusion protein was induced by IPTG. The histidine-tagged fusion protein was identified by Western blot and purified by Ni-NTA Agarose., Results: The recombinant plasmid PQE-30-SRG4 was constructed successfully and was expressed in E.coli M15. The expression of the fusion protein reached the top at 4-5 h after it was induced by IPTG. The fusion protein SRG4 with 6xHis tag was confirmed by Western blot and was purified by Ni-NTA Agarose., Conclusion: The recombinant plasmid PQE-30-SRG4 can be expressed in E.coli M15. The purified fusion protein including a Sad1_UNC like domain can be used for studying the biological function of SRG4 in spermatogenesis.

Aim: To construct the eukaryotic expression plasmid of insig2 gene and detect the expression of downstream gene adiponectin and adipocyte fatty acid-binding protein 2 (AP2) after the transfection of 3T3-L1 cells., Methods: Insig2 gene of the mouse was amplified by RT-PCR and then cloned into the eukaryon expression vector pEGFP-C(3), After confirmed by double restriction enzyme digestion analysis and DNA sequencing, pEGFP-C(3)-insig2 was transfected into 3T3-L1 cells by lipofectamine 2000. The expression of insig2 and downstream gene in the 3T3-L1 cells were detected by RT-PCR and fluorescence microscope., Results: The eukaryotic expression plasmid of pEGFP-C(3)-insig2 was constructed. The expression of fusion protein in the endochylema was confirmed. The transcription of adiponectin mRNA and AP2 mRNA was down-regulated after transfection for 24 h and 72 h., Conclusion: The eukaryotic expression plasmid of pEGFP-C(3)-insig2 is successfully constructed. The transfected insig2 may have an effect on fat metabolism of 3T3-L1 cells.