The Construction Of Efficient Expression System Of α And ε Toxins Of Clostridium Perfringens And Their Immune Protection Study

Clostridium perfringens is one of the main pathogen causing a variety of animalenterotoxemia, necrotizing enterocolitis, and human traumatic gas gangrene. C. perfringens isdivided into A, B, C, D, E type, and the exotoxins produced by the bacteria are the main causativeagents.The α and ε toxins generated by the type D C. perfringens cause animal enterotoxaemia, oreven death, and finally cost livestock industry substantial economic loss.So far, studies on α and ε toxins of the type D C. perfringens are very sparse.Therefore, in thepresent study, we firstly constructed two recombinant strains which can efficiently express α and εtoxins respectively; then the expressed α and ε toxin protein inclusion bodies were refined asantigens to immune rabbits; finally, according to change traits of antibody titers, we evaluated theprotection ability of the two expressed toxins.Firstly, the D-type standard strain of C. perfringens was cultured under anaerobic conditions,and then the genes of α andεtoxins were amplified by polymerase chain reaction (PCR) by meansof specific primers. On the basis of conventional recombinant DNA technology, the recycled PCRproducts (α andεtoxin gene fractions) were respectively inserted into expression vector pET28ato construct the recombinant plasmid. After identification through double-enzymecutting and DNAsequencing, the recombinant plasmids were respectively transformed into E. coli BL21(DE3), andthen optimal induction conditions of IPTG were explored. In order to detect the expressed proteinsspecificity, the products were subject to SDS-PAGE electrophoresis, nickel-affinity purification;PBS dialysis, and Western blot.200μg of purified toxin protein mixed with Freund adjuvant wasrespectively used to immune rabbits (α toxin: group A; εtoxin group B). The mixed toxins,together with Freund adjuvant were also used to immune rabbits (α andεtoxins: group C). Afterbeing periodically collected, rabbit sera were also analyzed by Western blotting and ELISA. Basedon the above results, the serum antibody changes in group A, B, and C were obtained.The results showed that,780bp fragment amplified by PCR was recognized as the α-toxingene by Blast alignment;972bp product amplified by PCR was also recognized as theε-toxingene by Blast alignment. Expression vector pET-28a and recombinant T vector containing thetarget gene were respectively subject to double-enzymecutting identification. Using molecularconnecting tecnique, we successfully constructed pET28a-α expression vector and vector pET28a-ε expression vector.After expression products were subject to SDS-PAGE electrophoresis, and then werespectively got the specfic proteins of28.9KD and36.9KD. The results of Western blot showedthat the recombinant proteins had a high specificity. After crude extracts of protein inclusionbodies were used to immune rabbits as antigens, antibody titres were monitored periodically byindirect ELISA. The results demonstrated that α-toxin antibody titre in group A reached maximumvalue5.7log2in the fouth week;ε-toxin antibody titre in group B achieved maximum value8.7log2in the fourth week; the antibody titre of α andεmixed toxins in group C achievedmaximum value4.7log2in the fourth week.The research results showed that the two recombinant strains BL21(DE3) could efficientlyexpress α and ε toxins respectively. The expressed toxins had a high immune protection whenbeing used to immune rabbits individually. The mixed toxins (α andε) also introduced a certainlevel of immune protection when being used to immune rabbits jointly.This research could providea theoretical basis and support for the further development of bivalent genetic engineeringvaccine.