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Expression and Purification Fusion Protein of a Novel Gene BC097361 Related to Angiotensin Ⅱ, Preparation and Identification of Its Polyclonal Antibody

Author: LiangZuoLei
Tutor: XingHuiChun；WeiHongShan；ChengJun
School: Shanxi Medical
Course: Infectious Diseases
Keywords: hepatic fibrosis HSC prokaryotic expression angiotensinⅡ BC097361 polyclonal antibody
CLC: R575.2
Type: Master's thesis
Year: 2008
Downloads: 34
Quote: 0
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Abstract


Objective:To produce a recombinant protein His-BC097361,which was fusionally expressed by in Escherichiacol.To prepare the BC097361 specific polyclonal antibody in rabbit and evaluate its immunoreactivity.Methods:1.BC097361 gene obtained from LX02 cell by reverse transcription polymerase chain reaction(RT-PCR) was inserted into pGEM-T cloning vector,then subcloned into inducible proeukaryotic expressive vector pET-32a(+).After confirmed by sequencing,the resultant vector was transformed into E.coli BL21(DE3).Fusion protein His-BC097361 was induced by IPTG and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting hybridization analysis.The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE,then was purified and renatured by Ni affinity column chromatography.2.The purified His-BC097361 fusion protein was used to immunize New Zealand rabbit to gain polyclonal antibody.The specificity and potency of polyclonal antibody was evaluated by Western blotting and ELISA.3.The expression of protein BC097361 in rat liver fibrosis tissues and in LX02 cells stimulated by angiotensinⅡwas detected with immunohistochemistry technique and Western blotting.Results:1.The BC097361 fusion protein was highly expressed and mainly was in inclusion body.2.The purified protein and its polyclonal antibody were obtained successfully.The titer of the polyclonal antibody,tested by ELISA,was more than 1:320 000.Its specificity and potency was confirmed by Western blotting and immunohistochemistry technique.3.The expression of BC097361 protein increased in the LX02 cells after treated with angiotensinⅡ36h and in in rat hepatic fibrosis tissues.Conclusion:The His-BC097361 fusion protein was successfully expressed and purified.The BC097361 specific polyclonal antibody was successfully prepared,which could supply a good tool for the study on the biological function of BC097361.The BC097361 protein was tested in the LX02 cells treated with angiotensinⅡand in rat hepatic fibrosis tissue.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis
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