Expression、purification Of β1 Integrin And Application Of Biopanning The Protein Combined With Helicobacter Pylori

Objective Hp is viewed as a facultative intracellularis bacteria in recent years, and it not only invades the cell, but also reproduces and produces toxins in the cell. Studies have confirmed that Hp can invade gastric cells by β1 integrin on the cell surface, but the bacterial protein related to this phenomenon and its effect are not clear. The purpose of this study is to obtain soluble β1 integrin protein through the prokaryotic expression and purification technology.Prepared β1 integrin protein biopans the Hp T7 phage display c DNA Library which is constructed by the laboratory, and the DNA sequence of Hp protein which interacted with β1 integrin is obtained. It will lay the foundation for studying the protein interacted with β1 integrin and its related function.Method ① CDS sequence of β1 integrin is obtained from GENBANK. The extracellular domain is retained and N terminal signal peptide sequence is truncated.② After analysing the distribution of β1 integrin rare codons, the rare condons of β1 integrin are optimized. According to the secondary structure of m RNA,the codons of TIR are adjusted. ③ The Total gene is synthesized and inserted into cloning vector of p UC18.The p UC18-β1-integrin plasmid and p ET30 a vector are digested by double enzymes respectively. Recycling DNA after Agarose gel electrophoresis and the DNA fragments are linked by T4 enzyme. ④p ET30a-β1-integrin plasmid is transformed into BL21 competent bacteria(DE3).Expression is induced after picking positive monoclonal. The expression is analysed by SDS-PAGE electrophoresis. ⑤ Inducing expression of the preserved strains, breaking strains by ultrasonic wave, then the supernatant is collected. Solubility of protein is analysed by SDS-PAGE electrophoresis.⑥ Inducing the expression of bacteria, collecting its inclusion bodies, and the liquid flow is taken through the Ni-NTA column.The protein is eluted by elution buffer which contains different concentrations of imidazole.Various elution buffers different in concentration are collected. The protein is analysed by SDS-PAGEelectrophoresis.⑦ Taking the soluble protein into a dialysis bag,using renaturation solution to dialysis three times, three times again by using storage solution,and filtering it through filters, the purified protein is obtained. The protein should be stored in refrigerator.⑧The Hp T7 phage display c DNA Library is biopanned by purified β1 integrin.Picking a single plaque to experiment with a PCR reaction,checking the reaction product by 1% agarose gel electrophoresis, recycling the reaction product of PCR,then it’s identified by sequencing technology which uses T7 up primer.⑨The obtained sequence is compared with homology sequence by Gen Bank database of NCBI.Results ①The result of single enzyme digestion of p UC18-β1-integrin plasmid is correct. ②The result of sequencing of p ET30a-β1-integrin plasmid which is bulit is correct.③p ET30a-β1-integrin plasmid is transformed into BL21 comp ETent bacteria(DE3), and expression is induced after picking positive monoclonal. The SDS-PAGE result shows that there is a zone near the molecular weight of 80 k D which belongs to β1-integrin.④ After inducing the expression of bacteria in 15℃,200 rpm,solubility of the protein is analysed by SDS-PAGE.The result shows that expression in the supernatant of β1-integrin is little, and it exists mainly in the form of inclusion body.⑤ Soluble β1 integrin is obtained by denaturation and renaturation of inclusion bodies.Its concentration is 0.32mg/ml. Using his-tag antibody to detect the target protein by Western blot, the result shows that there is a zone near the molecular weight of 80 k D. The result of experiment of protein freezed and thawed shows that there is not suspended matter after freezing and thawing,and also not precipitation after centrifugation.⑥Using prepared β1 integrin protein to biopan the Hp T7 phage display c DNA Library, 2 primary proteins which combined with β1 integrin are obtained after sequence alignment.Conclusion ① The soluble and pure protein of β1 integrin is obtained through the prokaryotic expression and purification technology.②Using β1 integrin protein to biopan the Hp T7 phage display c DNA Library,2 primary proteins which combined with β1 integrin are obtained.