The Expression Of Recombinant Bovine Prion Protein And The Production Of Monoclonal Antibodies Against Bovine Prion Protein

Prion diseases,also known as transmissible spongiform encephalopathies(TSEs),which are group of in animals and humans with fatal neuro-degeneration.Bovine spongiform encephalopathy(BSE),commomly called mad cow disease,is one type of TSEs in cattle that causes a spongy degeneration in the brain and spinal cord.The agent is a infectious proteins with null nucleic acid which is different substantially from viruses and bacteria.Prion diseases are characterized by accumulation of an abnormal(Pr Psc)form of a cellular prion protein(Pr Pc).At present,there is no effective treatment in prion disease,so strengthening the mad cow disease diagnosis is a main way to prevent the disease into China.The monoclonal antibody(m Ab)against prion proteins was not only used for mad cow disease diagnosis,but also was useful for treatment of mad cow disease that was the focus of recent researches.Objiective: The monoclonal antibody(m Ab)against mature prion proteins(Pr P)of bovine were prepared and on the basis of hybridoma technology,and the recombinant bovine prion proteins were regared as immunogen,and 6-week-old female prnp gene knockout mice(prnp-/-)were immunized in our study.Those m Abs were expected to establish a base for BSE immune research.Method: The E.coli BL21-p PROEX-htb-Bo Pr P of recombinant bacteria was activated,then sequenced.According to the Gene Bank,the result of sequencing was consistent with bovine Pr P amino acid sequence.The reombinant bacterium were induced by Isopropyl ?-D-thiogalactoside(IPTG),and their expressed products were identified by SDS-PAGE analysis.The fusion proteins was purified by Ni-NTA immunoaffinity chromatograghy.The inclusion body protein were pyrolysised by ultrasonic,then the recombinant expression products were renatured by dialysis and their protein concentration were measured by ultraviolet spectrophotometer.The reationgencity of fusion proteins were analysed by Western blot.The recombinant Pr P as positive immunogene and the E.coli BL21 as comparison immuogene were emulsified with Freunds complete adjuvant respectively.6-week-old female Prnp gene knockout mice(prnp-/-)were immunized by intraperitoneal injection,30?g per mouse.An indirect ELISA and Western blot detection method were established against anti rm Bov Pr P after the third immune,and the serum of immuned mice were detected by indirect ELISA and Western blot.Cell fusion was performed according to the traditional method.But beyond that,if the Pr Pc was expressed on the SP2/0 cells surface by Western blot before cell fusion.It was carried out to screen hybridoma cell line using recombinant bovine prion proteins as for the first kind of antigen by indirect ELISA assay,using the limiting dilution method,we finally obtained one hybridoma cell line which named 5C9D6.Its Ig type and subtype were determined by double-antibody layer ELISA.The specificity was analysis by Western blot.Result: We obtained a recombinant bovine prion proteins,and the proteins ultima concentration was 1?g/ul.An indirect ELISA detection method were established which packaged ELISA plate with 0.02?g/ml protein,5% skimmed milk as blocking agent,Incubated time both primary antibody and second antibody were 60 min.The immunity mouse serum titer was1:6400 using indirect ELISA.There was no expressed prpc in SP2/0 cells,so it could be used as cell fusion.The m Ab belongs to Ig G2 a.Western blot demonstrated that the m Ab 5C9D6 has stronger specificity,and responsed to the normal cellular prion protein of the wildtype mouse brain and the cattle brain.