| Hepatitis delta virus (HDV) is defective virus, its replication and spread depends on the presence of hepatitis B virus. HDV infection is usually associated with more severe liver damage, brings patients heavy physical and economic burden. HDV genome is a circular single-stranded RNA molecular of about 1.7Kb, and codes only one viral protein HDAg. The protein exists in two distinct forms:S-HDAg and L-HDAg. Except the increase of 19 amino acids in carboxyl end, other properties are the same, both of them can induce specific immunoreaction in the immune system. When infected with HDV, it induce different specific antibodies, IgM and IgG. HDAg is the only effective candidate antigens for serological diagnosis. Nowwe can express and purify bioactive recombinant HDAg on a large-scale by modern genetic recombination technology. Cooperation with the high density fermentation technology, not only we can get lots of bioactive protein, but also it makes combination of laboratory with market come true, it’s the important process for scientific research into the application.Based on the Escherichia coli codon preference, we recoded gene sequence of HDV antigen. GC% of update sequence is 51.2%, not contain repetitive sequences and the cis element that is not conducive to protein expression, e.g. SD similar sequences. We inserted improved sequence into expression vector pet43.la, and expressed a large number of recombinant protein in E.coli BL21 (DE3) under strong transcription ability of T7 polymerase, the protein amount can reach 23.6% of the total. Recombinant HDV antigen expressed in a soluble form containing good biological activities, meanwhile, we avoided refolding and other complicate operations. Application of high density fermentation technology to express recombinant HDV antigen, we explored many conditions, such as inoculation quantity、the carbon source、the nitrogen source、 the feedback feeding method、inducing way and inducing time during the fermentation. Finally, we determined the high density fed batch fermentation technology with carbon source as control factors. Under the condition of 5% inoculum size, we use glucose as control carbon source, control cell specific growth rate as 0.12h-1.In 1h using lactose as inducer, added 1mm IPTG and continued to induce for 3h. During induction, added 10g/L yeast powder to feeding culture. After the fed batch fermentation, the biomass OD600 absorbance can reach 62. After induction the recombinant protein accounted for 20% of the total bacterial protein. Through the direct relation between cells dry weight and protein,0.5g/l recombinant HDV antigen were obtained, has reached the standard of high cell density fermentation.Recombinant HDV antigen can be purified using immobilized metal ion affinity chromatography technology. Firstly, the bacteria were staved by ultrasonic method, and the impurities such as insoluble cell debris were removed by centrifugation under the condition of 12000g.Based on the properties that 6his-tag within recombinant HDV antigen combines specifically with metal ion of transition state, we did immobilized metal-chelated affinity chromatography depended on nickel ions. When used 0.5mol/L NaCl, metal ions cannot combine with his tag. With 1mol/L NaCl, metal ions can combine with recombinant HDV antigen. By changing the concentration of NaCl in the buffer solution, the purity the recombinant HDV antigen reach higher than 98%. High concentrations of salt is necessary for recombinant HDV antigen purification, the binding capacity of antigen and antibody increased significantly after treatment. The possible reason we speculate is that the high salt concentration changes the conformation of the recombinant HDV antigen, makes the conformation much closer to the native conformation which is beneficial to bind with antibody.We establish the indirect ELISA to measure IgG antibody in serum. Envelop ELISA plate by HDV recombinant antigen with 10 ng/well. Dilute serum in 10 folds, then add 50 μ L serum/well to pre-enveloped by HDV recombinant antigen plate, incubation in 37℃ for 30 minutes. After washing by PBST 5 times, add HRP labeled mouse anti human IgG antibody and incubation in 37℃ for 30 minutes. Add substrates A and B 50 μ L respectively then mix them. After 10 min add 1.8 mol/L sulfuric acid to stop reaction. Detect the OD in each well by using double 450/630 nm wavelength. In addition, we establish the capture ELISA to measure IgM antibody in serum. Add 50 u L serum/wells to pre-enveloped by mouse anti human IgM antibodies plate, incubation in 37℃ for 30 minute. After washing by PBST 5 times, add 100 u L HRP labeled HDV recombinant antigen/well. After incubation in 37℃ for 30 minutes washing by PBST 5 times. Add substrates A and B 50 u L respectively then mix them. After 10 min reaction detect the OD by the same method to indirect ELISA. We confirm the cut-off of two ELISA by1270 negative sera. In indirect and capture ELISA, OD<0.15 is negative and OD>0.15 is positive. The sensitivity and specificity of two ELISA both reached above 95% by 30 positive samples and 90 non-HDV sera. We establish the serology detection method depend on HDV recombinant antigen with high sensitivity and specificity.In conclusion, the experiment constructed recombinant prokaryotic expression vector of the HDV antigen successfully. In E.coli BL21(DE3) strains can efficiently express recombinant protein. We can get 0.5 g/L recombination HDV antigen by using high density cells fermentation technology. Changing the concentration of salt ions, recombinant antigen with both high purity and activity can be obtained by using affinity chromatography. HDV recombinant antigen ELISA kit meet the requirements of clinical medicine, applied by indirect and capture ELISA with HDV recombinant antigen to detect specific antibody in serum. We explore the technology which efficiently express recombinant antigen in E. coli by high density cells fermentation method. The technology is application valuable both in pharmaceutical industry and vitro diagnosis currently. The results have guiding significance for large-scale production and application of other proteins. |
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