| Ovine pulmonary adenocarcinoma(OPA), which can induce neoplastic transformation of alveolarand bronchiolar secretory epithelial cells, is caused by exogenous jaagsiekte sheepretrovirus(exJSRV).A clinical sign of OPA is the production of abundant frothy fluid in the lung,and drains when it lowers its head or during the‘wheelbarrow’test. Once the clinical signs areseen,the sheep will usually die in a few days or die abruptly following exercise or exposure tocold.OPA was first described in the19th century, ranging in age from2months to11years,andmost occur in aged2-4years,is a serious disease to the breeding sheep.There is no effectivemethods to control its spread,and therefore cause a great loss to the sheep breeding industry inmany countries around the world.The suitable cell lines which are stable and efficient for JSRV toreplication in vitro have not found yet,and also no JSRV pure virus was isolated,which hashindered the development of laboratory diagnostic and the research to the vaccines,specific drugand JSRV molecular biology characteristic.OPA is an important veterinary problem,in addition, itprovides a new model for molecular understanding of the epithelial cell tumours andHIV.Therefore it has a positive significance to establish a diagnostic method which are accurateand simple for the disease.A pair of special primers was designed using software primer premier5on the basis of the CAgene of JSRV. Subsequently, the CA gene was amplified by PCR, and then cloned into a pEASYplasmid for sequencing. The amplicon well sequenced was cloned into the vector pET28a+, leadingto the construction of a recombinant plasmid6CA66028al for protein expression. The rightrecombinant plasmid was initially induced using IPTG, and the expression products were analyzedby SDS-PAGE. After the expression condition was optimized, the correct recombinant protein waspurified and further used to immunize rabbits.The blocking ELISA method for the detection ofJSRV was preliminary established,which is based on using the pured recombinant protein asantigen,using the PcAbs as competitive antibody. Prokaryotic expression showed that expressionquantity was most when IPTG was0.4mmol/L,induction temperature was30degree,induction timewas5hours.A fused protein of28kDa in molecular was obtained, and existed in ultrasoundsupernatant, this also means that we had gotten the first anti-CA rabbit polyclonal which can combine with natural JSRV in China. In the western-blotting analysis, the rabbit antiserumrecognized the purified recombinant protein, as well as the JSRV extract, which should be helpfulin the establishment of the ELISA method.Blocking ELISA results showed that reaction conditionwas best when antigen concentration was2μg/ml,primary antibody dilution was1:2000, andblocked by incubation for1h with a1%(w/v)solution of bovine serum albumin in PBST, theantiserum and test samples were reacted first for45minutes as the resulting mixture,secondaryantibody dilution was1:1600and incubated for45minutes,colour development for12minutes.One purpose of the research was to understand the OPA infection situation of sheep samples,aswell as the actual clinical value of the ELISA method.In this research we collected23sheepsamples which was imported by Heilongjiang province from Australia,90sheep samples thatwere from9regions of the Heilongjiang province and120sheep samples that were from InnerMongolia where adjacent to Heilongjiang province for test.The results showed that lung samplespositive rate was61.11%,of which Inner Mongolia and Qiqihaer region infection rates were45.71%and46%respectively.The serum positive rate was40.83%,of which Inner Mongolia regionand heilongjiang province’s infection rates were52%and37.5%respectively, south west ofheilongjiang region infection rate was higher, the eastern region (hegang, jidong, jiamusi) and theAustralian imported sheep did not check out the cases of infection, the results was consistent withthe actual situation, which also indicated that the ELISA detection method is feasibility. |
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