Analysis, Purification, And Expression Of Recombinant Ricin Toxin A Chain (rRTA) And Its Mutant RTA (mRTA)

Ricin is a potent protein toxin, The holotoxin consists of two polypeptide chains (RTA and RTB) joined by a disulfide bond. RTA is a ribotoxin which inhibits protein synthesis in mammalian cells; RTB is a lectin which binds specifically to galactose residues on the cell surface and appears to trigger the endocytic uptake of ricin molecular. Because of its extraordinary toxicity and easy to be obtained, it is one of the important toxins in biological warfare agents and of highly hazardous bioterrorism agents. Therefore, it is of significance to perform an investigation into the recombinant RT antigenic fragments for developing diagnosis antibody and preventive vaccine.In order to reduce the toxicity and the probability of inducing vascular leak syndrome to human body, we designed three mutant RTA chains based on site-directed mutagenesis of key amino acid residues(D75,V76 involving in its ability to induce pulmonary vascular leak; Y80,Y123 and E177 involving in its active site) of RTA. The three mRTA chains were named mRTAD75AV76MY80A, mRTAD75AV76MY123A and mRTAD75AV76ME177D (abbreviatated to mRTAY80A, mRTAY123A, mRTAE177D in the following text). Moreover, hydrophilicity, antigenic index and surface probability of RTA and the three mRTA proteins were predicted by using the Protean procedure of Lasergene software package, the results showed that they had the same antigenicity.In this study, the plasmid pUC19-RTA was used as template to amplify a 822bp fragment encoding RTA gene by PCR and to generate the three mutant gene fragments (named mRTAY80A, mRTAY123A, mRTAE177D) by two times site-directed mutagenesis method. Then the target genes were individually ligated to the pMD18-T vector and the sequences were analyzed. The results indicated that the neucleotide sequences of RTA and the mRTA genes were entirely identical to the desighed requirements. The plasmids were named pMD18-TRTA, pMD18-TmRTAY80A, pMD18-TmRTAY123A, pMD18-TmRTAE177D, respectively. Subsequently, the resulting cloning plasmids and the prokaryotic vector pET-His were digested by the restriction endonucleases EcoR I and Nhe I. RTA and mRTA gene fragments were individually ligated to pET-His to construct four corresponding recombinant expression plasmids (pET-HisRTA pEJ-HismRTAY80A, pET-HismRTAY123A, pET-HismRTAE177D). After identified by PCR and EcoR I /Nhe I digesting, the resulting recombinant expression plasmids were individually transformed into E.coli BL21(DE3)plysS compotent cells and constructed four genetically recombinant strains (BL21/pET-HisRTA, BL21/pET-HismRTAY80A BL21/pET-HismRTAY123A and...