Cloning And Expression Of ScFv Gene For Anti-alpha-toxin And Its Biological Activity

SummaryCloning and Expression of ScFv Gene forAnti-alpha-toxin and Its Biological ActivityPh.D. Candidate:Zbao BaohuaSupervisor:Zbu PinClosa~ridiiitn Perfringer,s is ubquitous in the enviroment and has been found in soil, decaying organic matter and as a member of the gut flora in humans and animals.Different strains of C Perfringens can be assigned to one of five biotypes(A-E)depending on the spectrum of toxins produced. Biotype A strains are of particular importance as the aetiological agents of gas gangrene in humans. Gangrenous disease is of increasing significance in elderly and diabetic population,especialiy in those who have undergone lower limb surgery, where impaired blood supply to tissues can lead to anoxic conditions suitble for multiplication of bacteria. The disease can also arise in patients who have undergone surgery of the gaetrointestinal tract when contamination of damaged tissues with gut contents can result in the establishment of disease. A more periodic increase in the incidence of gas gangrene has occurred during armed conflicts when deep tissue wounds have become contaminated with soil. The failure to treat such injuries promptly resulted in the death of several hundred thousand soldiers during the First World War.In our experiment, the V11 and VL genes were amplified from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium Perfringens type A by RT-PCR. The V11 and VL genes were connected through a flexible linker (Gly4Ser)3 and the VH條inker梀L (ScFv) gene was cloned into a clone vector pGEM-T. The ScFv gene was sequenced and analyzed by computer. The ScFv-1A8 gene consists of 729 bp encording 343 amino acid residues. The ScFv-2E3 gene consists of 726 bp encording 342 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region gene segments. The variable region gene segments of ScFv-2E3 blong to mouse Ig heave chain subgroup II72(B and K light chain subgroup LII respectively.But The variable regi~in gene segments of ScFv-1A8 blong to mouse Ig heave chain subgroup 11(A) and K light chain subgroup VI respectively.Then, the ScFv genes were cleaved with Not I and Sfi I from plasmid pXScFv-2E3 and pXScFv-1A8 containing ScFv gene for anti-alpha-toxin of Clostridiurn Perfringens type A, isolated by 1.5% agarose gel electrophoresis, recovered the ScFv gene fragment. Then the ScF?gene with Nco I ans Barn LII was obtained by amplifing from the above ScFv segment. Finally these cleaved ScFv gene fragments were inserted into plasmid pHOG21, pET-20b, pET-28a, pUCI 19 which cleaved with Not I and Sfi I, Barn HI and Nco I respectively by cohesive-end ligation and expressed in JMTO9, XL 1-BLUE, BL21(DE3) respectively. After induced by IPTG,ELISA and SDS-PAGE analysis showed the ScFv could be expressed in the soluble periplasmic supernatant, culture medium and was produced in the form ofinclution body. The expressed Scfv was up to 4%,3%,O.9%,O.7% in the soluble periplasmic supernatant of recombinant strains XLL-BLUE(pHOG2E3), XLI-BLUE(pHOG-]AS),BL2 I (DE3)(pET2Ob-2E3) and BL2 1 (DE3) (pET2Ob- 1A8) respectiv ely. Especialy, the expressed Scfv-2E3 was highly produced up to 22% in the recombinant strain XL1-BLUE (pHOG-2E3), its molecular weight was 31 KD.At last, the expressed Scfv could neutralize phospholipase C activities of alpha-toxin and could provide protection for mice from the challenge of lethal dose of alpha-toxin of Clostridium perfrigens type A.The results showed the expressed Scfv had potential valuable use in theraping toxicosis of alpha-toxin.