Molecular Cloning And Prokaryotic Expression Of The Porcine IDâ…¡ And Selenoprotein S Genes And Preparations Of Polyclonal Antibodies

The purpose of this study is to obtain the gene sequence of IDⅡ and Sel S in the porcine, to express the IDⅡ and Sel S189gene from the pig lung successfully in Escherichia coli, to prepare rabbit antibody against porcine IDⅡ and Sel S by using IDⅡ and Sel S in the prokaryotic expressed in the prokaryotic expression system for the first time in China. The obtained antibodies are very useful in identifying both IDⅡ and Sel S in different tissues. The researches are significant for the investigation of physiological functions and action mechanisms of selenoproteins.In this study, the genes of IDⅡ and Sel S were successfully cloned through the classical molecular biologic approach from porcine lung tissue and expressed in E.coli BL21(DE3). After isolating and purifying the target proteins from the total proteins of E.coli BL21(DE3). Rabbit anti-IDⅡ and-Sel S polyclonal antibodies were raised with the purificating IDⅡ and Sel S proteins as immunogens. The obtained antibodies were used to detect the presence of IDⅡ and Sel S proteins in various tissues of porcine. Our existing theoretical and experimental research results can be informative for a further in-depth study of physiological functions of IDⅡ and Sel S. The main results and conclusions are summarized as follows:Firstly, a pair of specific primers is designed according to the published sequence of Sel S gene in NCBI GenBank. The total RNA was extracted from porcine lung tissue. cDNA fragments encoding Sel S were obtained with the specific primers by reversing transcription-polymerase chain reaction (RT-PCR) and cloned into plasmid. The results showed that the size of amplified Sel S was567bp. The sequencine analysis showed that the gene of Sel S was isolated successfully.Secondly, the TGA codon of IDⅡ, encoding selenocysteine residues was changed to TGT by site-directed mutagenesis.Thirdly, the sequence of the target gene was correctly inserted into pET-30a(+) vector to obtain plasmids pET-30a(+)-IDⅡTGT and pET-30a(+)-Sel S189. The DNA inserttion were confirmed by partial nucleotide sequencing. Prokaryotic expression plasmid, pET-30a(+)-IDⅡTGT and pET-30a(+)-Sel S189, were successfully delivered into E.coli BL21(DE3) by the calcium chloride method. Expectedly, the pET-30a(+)-ID ⅡTGT-fusion protein of～29.7KD and the pET-30a(+)-Sel S189fusion protein of～26.7KD were expressed, in transformants as revealed by the SDS-PAGE analysis.The results showed that the pET-30a(+)-IDⅡTGT-fusion protein and the pET-30a(+)-Sel S189fusion protein can be successfully expressed by the prokaryotic expression system.Fourthly, the anti-ID Ⅱ and anti-Sel S polyclonal antibodies were raised by immunizing rabbits with the purified target proteins by NTA-Ni affinity chromatography as immunogens. Antibody affinity was detected by indirect ELISA, and specificity of antibody was tested by western blot. The results showed that the specificity of polyclonal antibody, ID Ⅱ and Sel S, was good.Lastly, as determined by indirect ELISA analysis, ID Ⅱ and Sel S were detected in hypophysis, skeletal muscle, duodenum, liver and other organs. The results also showed that ID Ⅱ was found to be the highest in the hypothalamus and followed by skeletal muscle, duodenum and liver. The content of ID Ⅱ showed no much difference in the rest of the tissue. Sel S was found to be the highest in the skeletal muscle and followed by duodenum, liver and thyroid gland. The content showed no significant difference in the rest of the organs.In the conclusion, ID Ⅱ and Sel S were successfully cloned in this study. The prokaryotic expression vector were constructed, and the genes were successfully expressed in E.coil BL21(DE3). Purified ID Ⅱ and Sel S proteins were obtained and used to raise polyclonal antibodies. ELISA and western blot analyses showed that the antibodies displayed good specificity and sensitivity. The distribution of ID Ⅱ and Sel S was successfully determined in thirteen porcine tissue including hypophysis, skeletal muscle, duodenum and liver. All the research results provided a basis for further research on the distribution and function of selenoproteins.