| [Objective] Hepatitis D virus (HDV) can cause D type of viral Hepatitis. HDV virus nucleoprotein is the liver antigen, can stimulate the body to produce specific antibodies. Now some of laboratory using recombinant liver antigen as standard antigen, test the serm of patients. To obtain high purity of recombinant antigen is the first purpose of this experiment. And through the accumulation of data expression and purification we prepare for the next production. Currently there is no accurate data of the epidemiology of HDV in our country, mainly because of no unified inspection standard and reagent. Using the experimental production of HDV antigen establish the specificity and sensitivity are all better serological detection method is the second purpose of this experiment. Applied to the CDC work, get our country liver epidemiology of accurate data.[Materials and Methods]Through DNA recombinant technology, cloning and expression in E. coli protein, genetic engineering is a relatively mature technology. E. coli has a clear background, easy to operate, rapid growth and expression and high yield advantages, has been widely used serological detection reagent protein material acquisition. It can significantly reduce costs, and promote market-based reagents. In this experiment, the HDV sequence information obtained from Genbank database. Resequence the S-HDag HDV codon by making some codon substitution based on the preferred codon in E. coli. We predicted the secondary structure of the mRNA encodend HDV antigen, making some further optimization by affecting the expression of the codon sequences, then through artificial means to obtain the gene. By genetic engineering techniques, restriction enzyme digestion, then connect to the pET43. l.a expression vector in E. coli BL21(DE3) for expression engineering strain. By controlling the expression conditions, the final procedure for determining the expression:the standard procedures to obtain monoclonal and inoculated into a small test tube5ml,37℃overnight growth, then1:50dilution into250ml conical flasks at37℃until the OD value around of0.8, use ITPG as inducers, the final concentration of lmM/L, under37℃condtion induced about3h, finally the harvest is the soluble expression of HDV antigen. The medium used for the LB, optionally resistant to ampicillin at a final concentration of50mM/L.E. coli cells obtained using the ultrasonic disrupter, full of broken cells, centrifuged at10000rpm, the supernatant retained in1M/L of NaCl conditions, using the expression of the protein of His-tag. Competition between the nickel ions and imidazole is the basep of affinity chromatography on a protein capture, after the ion exchange chromatography and gel filtration, finally to obtain a high purity protein, it can accordance with standards of serological detection kit.The purpose of this experiment to obtain a high purity protein, in pH9.5carbonate buffer coated condition the protein in each hole is about2~4ng,4℃overnight using standard enzyme technology to horseradish peroxide enzyme-labeled anti-antibody detection kit assembled, from the liquidators of the small hepatic blood specimens and clinical serum samples collected for testing, the specificity and sensitivity of the method is good.[Conclusion]The protein with antigenic activity obtained from this experiment, and the method of serological assay has been established. Through genetic engineering, protein expression, we make preparations for further production. Cost advantages and good results for the detection methods can pave the market and to determine uniform standards and reagents, resulting in China’s accurate epidemiological data. |
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