小鼠骨桥蛋白的原核表达及多克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2015.05.005

Abstract

Abstract: In order to explore the function of osteopontin (OPN) regulated bone metabolism and the relationship between OPN and tumor, DNAMAN and Mega 5.02 softwares were used to analyze phylogenetic relationships of OPN protein, the OPN expression vector was prepared by RT-PCR and molecular clone; OPN was purified by SDS-PAGE gel slices, renatured by urea concentration gradient, and immunized mice to prepare the polyclonal antibody; The antibody titer and specificity were detected by ELISA and Western blotting, respectively.The homology comparison result of OPN sequence showed that it existed short repeat sequenc Arg-Gly-Asp (RGD) in different animals with evolution levels.Phylogenetic tree of OPN sequence showed that OPN had obvious evolution trend among animals.RNA was extracted, OPN recombinant vector had been digested and sequenced to confirm the correct construction, and strip lengths of OPN expression and purification were agreed with the prediction.The OPN antiserum titer was 1:1 600 detected by ELISA, and Western blotting proved that the antiserum had good specificity.We had successfully analyzed genetic evolution relationship of OPN sequence, gotten OPN expression vector, purified and renatured OPN, and prepared and identified the polyclonal antibody against OPN.

Key words: OPN protein; RT-PCR; polyclonal antibody; ELISA; Western blotting

CLC Number:

Q786

LIU Xiang, CHEN Chun-lin, WANG Yang-ke, JU Xiong, WU San-qiao, ZHANG Tao. Prokaryotic Expression,Polyclonal Antibody Preparation and Identification of Mouse Osteopontin Protein[J]. , 2015, 42(5): 1069-1075.

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Prokaryotic Expression,Polyclonal Antibody Preparation and Identification of Mouse Osteopontin Protein

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