Cloning, Expression, Purification Of Endogenous Angiogenesis Inhibitor Arresten Gene And Its Biological Activity Detection

Objective:In order to construct prokaryotic expression vector for endogenetic angiogenesis inhibitor Arresten gene and to optimize the expression condition, in the same time to investigate initially the anti-blood vessel activity of the expressing protein.Methods:According to the mRNA sequence and protein sequence of Arresten gene from the Genbank, specific primers for PCR were designed by using the software DNAman. The total RNA was extracted from a healthy puerpera's placenta organize of the normal puerperal, and human Arresten gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method, PCR production reclaimed and purified in low melting-point agarose and connected with plasmid pBV220. Molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220-Arr was identified by restriction enzyme digestion and sequenced. The pBV220-Arr was transformed into E.coli JM109 competent cell by CaCl2 transformation method and screening masculine colony in AMP table and evaluate in enzyme-cutting and PCR, in the same time to sequence. The pBV220-Arr was transformed into E.coli JM109,DH5α,BL21,BL21(DE3)and was expressed by this prokaryotic-expression system. The arresten expression level was detected by SDS-PAGE and was expressed high performance in the E.coli BL21(DE3). Through induction expression in temperature and time and dissolved oxygen, we determined a optimization expression system. The inclusion body containing target protein Arresten was separated and washed. The preliminary purified Arresten was dissolved in different concentration urea and renatured by gradient dilution. The expressed product was purified, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of CAM.Results:The Arresten gene was screening successfully, sequencing consequence indicates the difference of gene order on the 132 148 200 236 position. The recombinant plasmid pBV220-Arr was construted successful and the target protein Arresten can express as inclusion body in E.coli JM109,DH5α,BL21,BL21 (DE3), SDS-PAGE showed the expressed arresten protein was mainly inclusion bodies and had a molecular weight of 26 KD, The Arresten expression level of protein was highly in E. coli BL21 (DE3) and and the most optimization expression condition is that 30℃c ultivates 4h and 42℃induces to expresse 4h and .the dissolved oxygen content rate...