摘要
To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E.coli and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into E.coli DH5?for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E.coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from E.coli by a relatively simple method, which has high immunity activity.
To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E.coli and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into E.coli DH5?for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. E.coli cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E.coli accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from E.coli by a relatively simple method, which has high immunity activity.
基金
?73"Programme Funds(2001CB109001 and 2001CB109007)
"863"Programme Funds(2001AA212041,2001AA212291,and 2002AA212041).
