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Clone-expression and Polyclonal Antibody Preparation of Helicobacter Pylori ahpC Gene

Author: LiYanQing
Tutor: DuanGuangCai
School: Zhengzhou University
Course: Epidemiology and Biostatistics,
Keywords: Helicobacter pylori ahpC gene cloning gene expression
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 20
Quote: 0
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Abstract


Ahp(Alkyl hydroperoxide reductase) is one member of the peroxydase protein family,which is 23kD.These proteins play an important role in decomposition of hydrogen peroxide and hydrogen peroxide derivate.AhpC is composed of AhpF and AhpC.In recent study,Wang discovered that mutant strain defective in alkyl hydroperoxide reductase(AhpC) were more sensitive to oxidative stress conditions than wild type cells.And this ability to oxidative stress directly impact the living of H.pylori in the stomach of mice.AhpC gene sequences were obtained by PCR method.Construction of ahpC prokaryotic expression vector was carried out through design of suitable sites.Under optimized induction condition the positive clones efficiently expressed fusion protein. The fusion protein were purified by Ni ion affinity chromatography.Polyclonal antibodies were obtained in immunized rabbits.In the future studies we will further examine how the cagA gene impact on AhpC protein.Methods1.According to the multiple cloning site of vector pET30a(+),the primers were designed by using Premier 5.0 software.NdeI site was introduced in the upstream primer,and XhoI site was introduced in the downstream primer.2.The full-length ahpC was cloned by PCR from Hp27.The amplified DNA fragments were first ligated into pMD18-T vector then into pET-30a vector and last transformed into E.coli stain BL21.The positive clones were screened out by PCR.3.The positive clones were optimizated by adjusting induced concentration of IPTG and the induction time.4.Inclusion body was dissolved by using urea.The fusion proteins were purified by Ni ion affinity chromatography.5.Rabbits were immunized with fusion protein together with Freund’s adjuvant,and were immunized thereafter every a week with a total of 4 times.6.Antibody titers were detected by ELISA.Results1.PCR product showed that ahpC gene consists of 594 bp.It was cloned into pMD18T vector.2.The prokaryotic expression vector of ahpC was successfully constructed.The sequence homogenuity was up to 99%.3.The fusion could be expressed effeciently when it was induced by IPTG and the quantity was up to maxium when it was induced with 0.3 mmol/L IPTG for 5 hour. The purity of fusion protein was obtained by using Ni ion affinity chromatogrophy.4.High efficient and high specific polyclonal antibody was obtained by immunizing rabbits with fusion protein.Conclusions:1.Ni Affinity chromatography is an effective method for purifying protein with His-Tag.2.Immunizing rabbits with purified AhpC fusion protein can produce anti- AhpC fusion protein antibody,which can react with AhpC protein,thus this research lay foundation for further research.

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