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Staphylococcal Enterotoxins(SEs) is the major pathogenic factor of foodpoisoning and immunological diseases associated with Staphylococcus aureus,whichgrew into a health issue of the world.To date, more and more enterotoxin genetypeswere detected from different staphylococcus isolates, but lacking of effective proof onthe functions of their protein.However,for the constraint of traditional antibodypreparation technology, such as poor specificity of polyclonal antibody,largemolecular weight,weak affnity,high immunogenicity of monoclonal antibody,there isan adagissimo progress of in the detection for SEs and the treatment method forclinical illness caused by SEs.In this paper,by means of eukaryotic expressionsysterm,two genentically engineered recombinant antibodies against SEs weresuccessfully prepared,and the functional properties of these antibodies were putforaward as well.By using of improved TRIzol method, total RNA was extracted from sixhybridoma cells expressing Anti-SEs antibodies stably in order to the amplification oflight chain variable region gene(VL) and heavy chain varaible region gene(VH) byRT-PCR. The cloning and sequencing result shows that all the genes conform to thecharacter of classical variable region genes structure of mouse antibodies and belongto the funtional V(D)J gene reconstruction mode.Based on the homology analysis,sixVLgenes have an extremely high similarity in common,but VHgenes wereopposite.Some likely suppositions can be present that VHgene paly a more importantrole in the specific antigen recognition than VLgenes.Meanwhile, it is easier for SEsto induce the rearangement of VHgene to form the major antigen binding sites.On the other hand,ivs-IRES gene fragment and VLgene were spliced bySOE-PCR,which insert into the recombinant expression vector pcDNA3.1-VH tofinish the construcion of eukaryotic co-expression vector with single promotor,namedp2C2HILO,p5C12HILO. After the expression vector transfected into BHK-21cellsmediaed by Lipo2000,two mammalian cell lines that stably expresseddisulfied-stablized antibodies against SEs were finally constructed.By using ofEnzyme-Linked Immuno Sorbent Assay(ELISA), Indirect ImmunofluorescenceAssay(IFA) and Flow Cytometry(FCM) for immunoanalysis,both the two prepared recombinant antibodies have the same ability to bind to SEs,including natuaral SEA,SEA-his,SEG-his,SEK-his,SEO-his,SEQ-his,SEU-his.In addition,the new type ofAnti-SEs antibodies set out a potential application in the functional study, detection,prevention,diagnosis and treatment of SEs.
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