| Objective:Interferon-a is a broad-spectrum anti-viral and anti-tumor cytokines and also activate the immune system. Interleukin-2 plays an important role in anti-tumor, immunomodulation and the treatment of infectious diseases. It can also activate B lymphocytes, response to humoral immunity, promote the secretion of antibody, and enhance humoral immune responses. Mx proteins belong to the dynamin superfamily of large GTPase, and are induced by type I interferon in vivo.In this study, we transfected porcine IFN-a gene and interleukin-2 gene into eukaryotic cells.The gene expression and antiviral activity of the two proteins were detected. These results provide insights into the research and development of Vaccine adjuvants and the disease resistance breeding of transgenic animals. And The porcine Mxl promoter and Mxl genes were Successfully amplified and checked by sequencing.Methods:(1) According to the published sequence of porcine IFN-αand IL-2 genes,the two genes were amplified by RT-PCR, constructed to the eukaryotic expression vector plasmid pcDNA3.1,and respectively entitled pcDNA3.1-IFN and pcDNA3.1-IL;(2)The successfully constructed eukaryotic expression vector were transfected into eukaryotic cells. The cells integrated with the foreign genes were selected by G418. The integration of foreign genes were further tested by RT-PCR;(3)The mRNA of porcine IFN-αand IL-2 in eukaryotic cells were detected by Real-time quantitative PCR. The proteins expressed by IFN-a and IL-2 gene in eukaryotic cells were detected by ELISA. The biological activity of the two proteins was detected by lymphocyte proliferation test (MTT).(4) According to the published sequence of porcine Mxl promoter and Mxl genes,the two genes were amplified by RT-PCR, constructed to the eukaryotic expression vector plasmid PGKneotpALox2.Results:(1)The porcine IFN-a and IL-2 were Successfully amplified and checked by sequencing. The PCR fragments were inserted into eukaryotic expression vector pcDNA3.1 and the plasmids pcDNA3.1-IFN,pcDNA3.1-IL were constructed successfully.(2)The eukaryotic cells in which the porcine IFN-a and IL-2 genes expressed stably were selected by G418 and the further identification by RT-PCR was positive.(3) By Real-time quantitative PCR we found that the expression level of IFN-a mRNA in the cells into which the IFN-a gene was transfected was higher than the control group (untransfected group) by 1.52 times; the expression level of IL-2 mRNA in the cells into which the IL-2gene was transfected was higher than the control group (untransfected group) by 1.43 times. The detection of cells (integreted with foreige genes) supernatant by ELISA showed that the IFN-a is 121.45 pg/mL.in the control group (untransfected group) IFN-a is 89.36 pg/mL which is lower than the transfected group by 1.35 times; IL-2 is 97.45pg/mL which is higher than the control group(75.36 pg/mL)by 1.29 times. The statistical software SPSS analysis showed that the lymphocyte proliferation of supernatant of the eukaryotic cells integrated with foreign genes was significantly different negative control group, these results indicated that the products of these two genes have good activity, and can be used to resist the virus.(4)The porcine Mxl promoter and Mxl genes were Successfully amplified and checked by sequencing. The PCR fragments were inserted into eukaryotic expression vector PGKneotpALox2.Conclusion:Porcine IFN-αand IL-2 gene can express successfully in eukaryotic cells, and the proteins have good activity. These results provide insights into the research and development of Vaccine adjuvants and the disease resistance breeding of transgenic animals. The porcine Mxl promoter and Mxl genes were cloned from pig lymphocyte by RT-PCR and ligated into PGKneotpALox2 vector,... |
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