Specific Gene Of Trichinella Spiralis Adult Worm CDNA Library Construction And PCNA Cloning, Expression And Functional Identification

Trichinosis pathogens is spiral brush nematodes (Trichinellq spiralis)referred to as "trichina, parasitic in swine, wild boar, rat, bears and other150kinds of animals and humans, imagoes and larvae are parasitic in the samehost within the small intestine. Trichinosis is raw or half feed containingtrichinella spiralis larvae capsule package of pork and other meat and animalinfection, the disease is more common in the world of beast comorbidities.Not only cause serious damage to human health, and to the pig industry cancause huge losses. In recent years in17provinces, municipalities andautonomous regions in China have found several human outbreak of infection.For our country in henan province trichinosis infections, one of only sevenoutbreak has occurred, zhengzhou city, the onset of more than400people. Ifnot timely diagnosis and treatment, patients can be as high as3%a30%mortality rate. Thus trichina become the meat hygiene inspection of firstparasites, trichinosis of prevention and control work more urgency andimportance in our country, the key of prevention and control of trichinosis isto establish a rapid, simple and high specificity of detection means, and takevarious effective prevention measures to reduce animal infections.Oftrichinosis immune prevention study, as a result of trichinella spiralis indifferent development period of the worm antigen components exist obviousdifferences and in the same issue worm antigen structure is extremely complex,and in its long-term parasitic life, gradually formed the immune escapemechanism, so currently used component complex worm antigen is oftendifficult to obtain ideal immune protective effect. Trichinella antigen has itsspecificity, composition is extremely complex, and cannot be a large number of in vitro cultivation and breeding, and its clinical manifestation is relativelycomplex, so bring difficulties to trichinosis immune diagnosis and prevention,so looking for trichinella spiralis specific antigen gene has become the focusof our work. Hook and construct a cDNA library, immunological methodscDNA fragments, thus analysis of antigen epitope antigen protein, selectingthe best antigen, is one of the effective ways to study trichinella spiralisantibody.Objective:Collect pure five trichinella spiralis adult day age,to complete the cDNALibrary of Trichinella spiralis and cloning and sequencing of Trichinellaspiralis PCNA antigen.Then log on PCNA in the GenBank sequencecomparison. Constructing trichinella spiralis proliferating cell nuclearantigen (pcna) gene prokaryotic expression vector, induced the expresspurpose protein and westen appraisal its function.Methods:Collect pure trichinella spiralis imago.According to GenBank in thelogin sequence of proliferating cell nuclear antigen gene of Trichinella spiralisdesign primers,using PCR method from the gene pool of adult Trichinellaspiralis spoofed this gene, resulting PCR product and pUM-T carrierconnection.Conversion to a competent DH5a.The sequence results wereanalysized with BLAST software. Through the BamHI and HindII doubleenzyme and product recovery ways to get expression vector, so as to constructthe expression of recombinant, will build a good expressive recombinant intocompetent BL21. Through the IPTG induced express purpose protein, thewesten appraisal its function.Results:1Extraction trichinella spiralis imago RNA:In the glue can see clearlythat the28s,18s,5s three all roads belt, banding brightness is good, nosignificant degradation phenomenon that the integrity of the RNA is good, canbe cDNA synthesis, and can be used in the library construction.Andelectrophoresis images found that trichinella spiralis18s rRNA banding brightness is obviously higher than that of the28s rRNA banding brightness,about2times, after repeated repeat, results are consistent.2Constructing trichinella spiralis imago cDNA library：After two hybridand two PCR, the cDNA library for electrophoresis analysis got mass banding.3The expansion of PCNA gene：The expansion of PCNA gene, its size isabout804bp. Cloning genes and GenBank login proliferating cell nuclearantigen (pcna) gene homology is94%.4Expression vector construction: use BamHI and HindIII to expressionvector pET32a, enzyme and product recovery, obtained the expression vectorconstruction.5PET32a-PCNA recombination expression plasmid appraisal: withrestriction enzyme Bam H I and Hind III double enzyme after recombinantplasmid get2500bp and804bp or so two segments, and expected resultsconform to, show that pET32a-PCNA prokaryotic expression vectorconstruction success.6Recombination expression plasmid pET32a-PCNA expression: expressproduct for sds-page, in30kD position can be obviously in the protein band.7Western blot do function identification: with infected with the trichinellaspiralis in mice serum do one resistant, get obvious banding, and identified theantigenicity PCNA.Conclusions:1Complete trichinella spiralis imago cDNA library establishedTheproliferating cell nuclear antigen is successfully cloned.2Successful constructs the yunnan strain trichinella spiralis proliferatingcell nuclear antigen (pcna) gene prokaryotic expression vector.3Successful expressed PCNA.4With the method of Western successful proliferating cell nuclear antigen(pcna) verified the antigenicity.