Purification of Dunaliella salina glucose-6-phosphate isomerase (GPI)recombinant proteins in engineering bacteria and preparation of its polyclonal antibody
The purpose of the present study was to purify Dunaliella salina glucose-6-phosphate isomerase (DsGPI) recombinant proteins from engineering bacteria BL21(DE3) and prepare its polyclonal antibody. The DsGPI gene was cloned by PCR using primers with Nde Ⅰ and EcoR Ⅰ restriction enzyme cutting sites and then inserted into the Nde Ⅰ-EcoR Ⅰ sites of the prokaryotic expression vector pET28a ( + ) to create a recombinant plasmid pET28a-DsGPI. After the competent E. coli BL21 (DE3) cells were transfected with the pET28a-DsGPI, the total proteins from E. coli BL21(DE3) with pET28a-DsGPI were analyzed by SDS-PAGE. The inclusion bodies of the DsGPI recombinant proteins were washed by urea buffer to eliminate the unwanted proteins. The recombinant proteins were dissolved by soluble buffer with 8 M urea and then purified by Ni-His tag column. After the purified proteins were used to immunize 2 New Zealand rabbits, the specificity and sensitivity of the prepared anti-sera were characterized by Western blots and indirect ELISA, respectively. The results showed that the DsGPI gene was expressed effectively in E. coli BL21 (DE3) with the recombinant proteins being up to 41. 6％ of the total expressed proteins, and that the anti-sera from the immunized New Zealand rabbits bound specially to target proteins from D. salina cells by Western blots and the titer of anti-sera was 1:512 000 examined by indirect ELISA. It is concluded that polyclonal antibody against DsGPI with high specificity and sensitivity has been prepared using the purified DsGPI fusion proteins as antigens. The prepared antisera to DsGPI makes it possible for studying the expression of the proteins in D. salina.
glucose-6-phosphate isomerase prokaryotic expression polyclonal antibody
Lei Zhang Liuqing Cui Jie Li HongtaoLiu Lexun Xue
Laboratory for Cell Biology, Department of Biology, Zhengzhou University, Zhengzhou, 450001, China
国际会议
南京
英文
217-220
2010-05-12（万方平台首次上网日期，不代表论文的发表时间）