RESUMO
AIM: To clone and express human alanine aminotransferase 2 (ALT2) in E.coli Rosetta (DE3), and to prepare monoclonal antibodies(mAb) against ALT2 for diagnostic purpose. METHODS: The gene encoding alanine aminotransferase 2 (ALT2) was cloned from hepatoma carcinoma cell by RT-PCR, and then inserted into pET28a vector. Recombination plasmids (pET28a-ALT2) were transformed into E.coli BL21. Human ALT2 was expressed as His-tagged fusion proteins and purified by immobilized Ni(2+);-affinity chromatography. The purified fusion ALT2 protein was used as an antigen to prepare mAb against it. RESULTS: The fusion ALT2 protein was expressed in recombinant E.coli Rosetta (DE3). The enzymatic activity of purified His-tag ALT2 is over 10 000 U/L. Mice were immunized with the purified fusion ALT2 protein, and 5 mAbs against ALT2 were generated. CONCLUSION: Two mAbs with high specificity for ALT2 were selected for further quantitative diagnostic reagent development.
Assuntos
Alanina Transaminase/genética , Alanina Transaminase/imunologia , Anticorpos Monoclonais/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alanina Transaminase/biossíntese , Alanina Transaminase/isolamento & purificação , Animais , Especificidade de Anticorpos , Linhagem Celular , Escherichia coli/genética , Expressão Gênica , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Camundongos , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
BACKGROUND: Seroprevalence data suggest that a third of the world's population has been infected with the hepatitis E virus. Our aim was to assess efficacy and safety of a recombinant hepatitis E vaccine, HEV 239 (Hecolin; Xiamen Innovax Biotech, Xiamen, China) in a randomised, double-blind, placebo-controlled, phase 3 trial. METHODS: Healthy adults aged 16-65 years in, Jiangsu Province, China were randomly assigned in a 1:1 ratio to receive three doses of HEV 239 (30 microg of purified recombinant hepatitis E antigen adsorbed to 0.8 mg aluminium hydroxide suspended in 0.5 mL buffered saline) or placebo (hepatitis B vaccine) given intramuscularly at 0, 1, and 6 months. Randomisation was done by computer-generated permuted blocks and stratified by age and sex. Participants were followed up for 19 months. The primary endpoint was prevention of hepatitis E during 12 months from the 31st day after the third dose. Analysis was based on participants who received all three doses per protocol. Study participants, care givers, and investigators were all masked to group and vaccine assignments. This trial is registered with ClinicalTrials.gov, number NCT01014845. FINDINGS: 11,165 of the trial participants were tested for hepatitis E virus IgG, of which 5285 (47%) were seropositive for hepatitis E virus. Participants were randomly assigned to vaccine (n=56,302) or placebo (n=56,302). 48,693 (86%) participants in the vaccine group and 48,663 participants (86%) in the placebo group received three vaccine doses and were included in the primary efficacy analysis. During the 12 months after 30 days from receipt of the third dose 15 per-protocol participants in the placebo group developed hepatitis E compared with none in the vaccine group. Vaccine efficacy after three doses was 100.0% (95% CI 72.1-100.0). Adverse effects attributable to the vaccine were few and mild. No vaccination-related serious adverse event was noted. INTERPRETATION: HEV 239 is well tolerated and effective in the prevention of hepatitis E in the general population in China, including both men and women age 16-65 years. FUNDING: Chinese National High-tech R&D Programme (863 programme), Chinese National Key Technologies R&D Programme, Chinese National Science Fund for Distinguished Young Scholars, Fujian Provincial Department of Sciences and Technology, Xiamen Science and Technology Bureau, and Fujian Provincial Science Fund for Distinguished Young Scholars.
Assuntos
Vírus da Hepatite E , Hepatite E/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/imunologia , Adolescente , Adulto , Idoso , China , Método Duplo-Cego , Feminino , Hepatite E/imunologia , Vírus da Hepatite E/efeitos dos fármacos , Vírus da Hepatite E/imunologia , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.