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Prokaryotic and Eukaryotic Expression of Human Gad Gene and Preparation of Its Polyclonal Antibody

Author: AnZuo
Tutor: XuXia
School: Guangzhou Medical College
Course: Immunology
Keywords: gAd baculovirus insect cell sf9 prokaryotic expression polyclonal antibody
CLC: R341
Type: Master's thesis
Year: 2010
Downloads: 25
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Abstract

Research ObjectiveThe bac to bac system was used for expressing bioactive gAd protein , and then study the interaction mechanisms between gAd and adiposity, diabetes, CVD, eclampsia,and etc. Building prokaryotic expression vector PET-28a(+)-gAd for gAd gene , preparing polyclonal antibody, then the result make the groundwork for further researching in clinical examinations of adiponectin’s concentration in human serum.Research Methods(1)The first part Amplified human gAd gene by PCR method using human genome as a template,insert the gene to plasmid of pFastBacHTB and transpose into DH10Bac containing a bacmid shuttle vector. Selected successful transposons containing Bacmid-gAd by screening,then transfected Sf9 cells with the obtained bacmid-gAd plasmid in the mediation of liposome.Next,identified the expressed product by SDS-PAGE and western-blot.(2)The second part Amplified human gAd gene by PCR method using human genome as a template,to constructed prokaryotic expression vector PET-28a(+)-gAd and transposed it into BL21(DE3),After inducing its expression by IPTG, the expression product was checked by SDS-PAGE and western-blot, then purified the target protein by Ni2+ affinity column. Finally,the purified protein was used as the antigen to immune the New Zealand White rabbits for preparing the polyclonal antibody.Research Results(1)Sf9 cells infected by the recombinant baculovirus was observed to have obvious morphological changes. SDS-PAGE profile showed a new protein band with a relative molecular weight of 15～25KD, and western-blot proved that it can combine with the anti-His antibody.(2)The expression protein was obtained from the BL21(DE3)which has been transposed by PET-28a(+)-gAd vector by IPTG. Western-blot proved that the expression protein can combine with the anti-his antibody. Furthermore, there is only one strip after electrophoresis analysis of the protein that had been purificated. The titer of polyclonal antibody obtained from the immunized rabbits was over 1:32 000 by the methods of ABC-ELISA .Western-bolt confirmed that antiserum has specific recognition to combine with gAd protein.Conclusion(1) Human gAd gene was successfully expressed in Sf9 cells, which lay the foundation for further research the mechanism of adiponectin in the development of some diseases such as obesity, insulin resistance and type 2 diabetes mellitus as well as its therapeutic value in these diseases.(2)Prokaryotic expression vector PET-28a(+)-gAd was constructed successfully. The expression protein has been obtained and purified to prepare polyclonal antibody, and the antiserum has high titer and specificity,which lay the foundation for further preparation of gAd monoclonal antibody . Also, the result will make the groundwork for further researching in clinical examinations of adiponectin’s concentration in human serum.

Prokaryotic and Eukaryotic Expression of Human Gad Gene and Preparation of Its Polyclonal Antibody

Abstract

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