Cloning And Expression Of Human And Mouse CR1 Gene In E.coli And Pichia Pastoris As Well As Polyclonal Antibodies Preparation

The h-CR1 ( Human Cell Regulator 1, GenBank accession number AC021016) is a novel human gene cloned in the lab of Deptof Pathway Engineering at the Institute of Medicinal Biotechnology. The yeast two-hybrid screening (using the human skeleton muscle cDNA as library) indicated that h-CR1 is related with the proteins involved in regulation of muscle contraction and cell signal transduction. Online protein prediction analysis showed that there is a transmembrane region in this protein, and there were homologous gene sequences in mouse, rat, cattle and pig genome, but none in the yeast and fly genome. This suggested that CR1 gene would only present in mammalian genome.Primers were designed and synthesized through bioinformatics analysis, total RNA was extracted from the mouse C57BL/6J spleen cells, then a cDNA of a 0.5kb DNA fragment was obtained by RT-PCR technique. Sequence analysis indicated that it contains a complete ORF, named m-CRl (GenBank accession number AY299972), coding 142 amino acids which shows 90.1% identity with the known h-CRl protein. Blast Search revealed that there were 13 amino acids differences between m-CRl and h-CRl protein, among them 10 were homo-amino acids changes, proved that the h-CRl and m-CRl are highly conserved.Through in vitro recombination with plasmids pPIC9 and pET24a(+), several of h-CRl gene express vectors in Pichia pastoris GS115 and E.coli BL21-CodonPlus(DE3)-RIL system were constructed. An integrated and secrete expression system for h-CRl gene in Pichia pastoris was established. The effect of N-terminal and/or C-terminal tag fused with h-CRl gene on the expression of h-CR1 gene in E.coli system was compared. The result showed that h-CRl protein expressed as inclusion body with high efficiency when it is fused with N-terminal T7-Tag. This strategy was also applied to express m-CR1 protein successfully in E.coli.According to the physico-chemical character of CR1 as a membrane protein, Tag-hCRl and Tag-mCRl fusing proteins were purified with several following steps. The solution of re-natured inclusion body was dialyzed and precipitation was collected, target proteins were separated by preparative SDS-PAGE electrophoresis, and electric elution then applied to recovery the proteins. The concentration of purified proteins were 2mg/ml with the purity about 95%.Purified proteins were used to immunize rabbits and the anti-Tag-hCR1 and anti-Tag-mCRl polyclonal antibodies were prepared. The titer of the prepared antiserums were about 1:105 with high immune specificity, determined by single immune-diffusion test, ELISA and Western Blot. This work made it possible to further study the biological function of CR1 gene with immunological and cytobiological methods.