Study Of Construction And Expression Of Recombinant Human Collagen Type â…¡ Peptide 250-270

Rheumatoid arthritis ( RA ) is an autoimmune disease. Type II collagen (CII ) is a signification autoantigen associated with the development and theraphy of RA. The high-level-expression and purification of recombinant human collagen type II peptide 250-270 (rhCII250-270)are the purpose of the present study, which provides the material for basal and clinical reaserch of RA. Adopting the methods of DNA synthesis, polymerase chain react ion (PCR), recombinat DNA technology, DNA sequence and affinity chromatography, we construct the poly-linked-genes of rhCII250-270 successfully, study the expressive conditions of that in escherichia coli(E. coli), and purify the expressive production.To express rhC II250-270 in E. coli effectively, we adopt some methods for destination gene to optimize. The gene of rhCII250-270 was constructed by the methods of DNA systhesis chemically and PCR adopted the preferable codes in E. coli. The gene was recombined intopUC19 plasmid and sequenced. The sequencing result is conform with the designed. The poly-linked-gene was constructed by signal-genes in recombinant plasmid using the same syicky tips digested by BamHI and Bglll.The outcome of sequencing is consistent with the expected.In study of expression, we compared the expressive production amounts of rhCII250-270 among in different expressive vectors and in different E. coli. The amounts of nonfused expression which induced either chemically or temperature control, were lower than that of fused expression in various of E. coli. The solubility of expressive production is the same result. The optimized conditions which of fused expression in E. coli BL21 was studied. SDS-PAGE indicated that the size of expressive production is 43kD which is accord with expected, the amount of that is 30% of the total bacterial protein, and the solubility is above 80%. GST affinity chromatography was used for purification of expressive production. The degree of purity for purified protein is above 90%.In the present study, the human immunodomnimant peptide of CII was recombined, expressed and purified for the first time. The exploration for the high level expressive conditions of collagen in procaryotic cells, should afford some experience for the same study lately. The present study could also provides the material for basal and clinical reaserch of RA.