OBJECTIVE: To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification...OBJECTIVE: To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system. METHODS: Hepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis. RESULTS: Cloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L. CONCLUSION: 25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.展开更多
Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reactio...Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology展开更多
基金TheworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina (No 3 963 0 2 80 )
文摘OBJECTIVE: To investigate the role of the 25 kD hepatitis B e antigen (HBeAg) precursor that only exist inside hepatocytes and study its effect on the pathopoiesis of hepatitis B and QIAGEN expression and purification system. METHODS: Hepatitis B virus (HBV) preC/C gene for the 25 kD HBeAg precursor was cloned into the expression vector pQE30 and the 25 kD HBeAg precursor was expressed in Escherichia coli (E. coli) and purified. Its antigenicity for 21 kD mature HBeAg was tested by western blot analysis. RESULTS: Cloned fragments in the expression vector were sequenced and verified to be homogeneous with that of HBV (ayw subtype). Expression of the HBeAg precursor in E. coli under the transcriptional regulation of T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 25 kD. Recombinant HBeAg precursor exhibited identical potencies with 21 kD mature HBeAg that reacted with anti-HBeAg antibodies. The purification rate of the expressed HBeAg precursor was up to 89.6% and the yield of purified HBeAg precursor from this procedure was 2.4 mg/L. CONCLUSION: 25 kD HBeAg precursor exhibited biological activity and might play an important role in pathopoiesis of hepatitis B.
文摘Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology
