| Hemorrhagic fever with renal syndrome( HFRS) is a acute human communicable disease caused by some type Hantaan virus of the Bun-yanviridae, and is characterized with fever, hemorrhage and acute renal impair,Clinically. A traditional laboratory diagnostic method such as indirect immunoflourescent antibody assay (IFAT) difficulty satisfies convenient and accurate request because of the artificial operative errors and complex trials. We adopted the technique of the gene engineering, clones and express S gene in the colibacillus obtained sufficient particular core protein which was used as a antigen for coating board of enzyme labeling. We set up a indirect ELJSA which was used for detecting special IgG antibody in patient sera for assisting the diagnosis of HFRS, clinically. In the same time we detected the antibody levels of the healthy population at Shenyang regeion as well as thirsty cases, satisfactory results were obtained.MethodsThe receptor bacillus is E col ;JM 109, expressed carrier is PGEX-6p - 1. We designed special primer, ourselves using plasmid PAC -Hans S as template for amplifying S gene. Using double enzyme cutpalsmid PGEX - 6p - 1 and S genetic fragment diffused by BglllandXhol double enzyme cut. DNA was recombinated in vitro under theeffect of ligase. The transformated receptor E col:jM109 of recombinat-ed DNA was screened by drug plate recombinated strain was obtained by extracting strain plasraid and electrophoresis detection. Further atlas of plasmid enzyme cut was identified, amplifying by PCR was carried out,thus recombinated E col:jM109pGEX -6p - 1 - HansS strain was obtained. The obtained strain was cultured with inducing of IPIG. Thal-lus was obtained, destroyed it with low temperature ultrasound, collected upper pure fluid. SDS - PAGE electrophoresis was carried out. Protein board expressed the quantity of special S gene was obtained. Upper puer fluid was ability and chromagraphy. Using confluent protein as antigen enzyme - labeled plate was coated, positive sera and immune western blot were detected with indirect ELISA, identifying the S confluent Protein. One hundred serous samples from healthy population were derected with indiredt ELISA, obtained the average value of sera of healthy population, serous samples of thirty cases were also detected , both values were compared with IFAT slide method, satisfactory results were obtained.Results1. Recombinated gene engineering bacillus E col;jM109pGEX -6p - 1 - Hans. 2. Special 73KDs confluent protein was obtained from avidity, chromagraphy and pureness.3. Indirect ELISA was set up detecting antibody of healthy population at Shenyang region and of special antibody in the patients with HFRS. Through the comparison with clinical feature and IFAT slide method, satisfactory results were obtained.Discussion1. A construct of double enzyme cut was applied for recombinat-ing plasmid. Enzymes of BamHI and Xhol were used as carrier. The fragment was amplified with method of PCR. Because of the difference of two recombinated cohesive ends, advantaging a correct insertion into the fragment,it is advantageous to selecting recombinating clone. But because of existing BamHI cutting point in the S genectic region which may cutting off the amplified S gene, complete S protein cant be obtained. Because of no existing Bglll cut point in gene S, therefore we used enzyme Bglll replacing enzyme BmHI , though cognition site of both enzyme Bgl H and BamHI and different, BamHI at GGATCC while , Bgl If at AGATCC, but both enzyme may produce similar end GATC thus both their actual end may link cohensive end each other. Update no reports were seen in references about our design in the clone expression of HFRS S gene.2. We selectively used confluent expressive pronucleus carrier, that is, character of ST expressive system is using glutathione S tran-ferase of confluent protein, not only elevating expression of solubility of extrinsic protein but alse elevating special method of detection and pureness. Such carrier carries p...
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