Expression, Purification And Cytotoxicity Of Recombinant Immunotoxin ScFv-PE40 In E.coli

Cysticercosis cellulosae is one of the most important Znonotic Parasitic diseases in our country. It causes great economic losses, and has serious effect on public health. Human is not only a intermediate host but also a terminal host. Cysticercus is a kind of parasite which often lives in manyorgans such as brain, eyes ,heart and so on .When living innervous system ,it can cause serious disease ,for example cerebral cysticercosis which does great harm to health of human. Since many years ago , prevention and cure of this disease was by means of drugs. But because of drug-fast and drug residue, immuno-prophylaxised cysticercosis cellulosae was more important than drugs cure, and it was well known in parasite educational circles. our study-group had obtain McAb of against Cysticercus Cellulosae Scolex antigen, and had extract single-chain antibody ScFv from it, and had linked with Pseudomonas exotoxin PE40, then cloned to plasmid vector pET28a. The aim of our present study was to express and purify recombinant immunotoxin ScFv-PE40,and then carry out oncosphere kill and wound experiment for detect recombinant immunotoxin effect. This experiment was the basis of animal experiment and recombinant immunotoxin applied and was the theory of prevention and cure Cysticercosis cellulosae.Methods:.First,Competence E.coli BL21 was made by CaCl₂. Then, pET28a which contain single-chain antibody gene (ScFv) and Pseudomonas exotoxin gene (PE40) was transformed to E.coli BL21. After cultivation, bacterio-fluid was spread on solid culture media. Bacterio-fluid was stayed over night in 37℃and observed next day. From it, 12 bacterial colonies was obtained and then cultivated. After it, the plasmid was extracted. The result was identified by EcoR I and Hind III enzyme digestion. Bacterial colony was inducted expression. The result analysised by SDS-PAGE and Western-Blotting to detect the activity of protein. because of expression vector pET28a(+) which contain His-Tag in N tip of fusion protein,protein was purified by Ni²⁺ affinity column.Then,through vitro oncosphere kill and wound experiment to detect the effect of killed Cysticercus by interest protein and draw kill and wound curve figs at the same time. The research results and expression product could serve as a basis for further studies on toxin immunity effectiveness,and for prevention and treatment of cysticercus disease at the same time.Result: After the recombinant plasmid which contain pET28-ScFv-PE40 was conversed to E.coli BL21,plasmid was extracted and identified by enzyme digestion of EcoR I and Hind III. Through analysis by agarose gel electrophoresis,there were two nucleic acid band which located 5400bp and 1800bp. the nucleic acid length was samed as sequence length of determination before. The result indicated the plasmid was the purpose plasmid. E.coli BL21(DE3) was inducted expression by IPTG. The expression product was analysis by SDS-PAGE. The result indicated that ScFv-PE40 gene had expressied and they were no different among every expression and the relative molecular weight of purpose protein was 68kDa(pET-28a was 3.5 kDa and ScFv-PE40 was 68 kDa).This result was same as ScFv-PE40 expressive protein and expressive dose was account 13.8%.But the control group which contain vacancy plasmid pET-28a did not observed the same expressive protein. To further indicated the expressive things,we made the protein electrotransfer to the PVDF transfer membranes. The first antibody was Mus-anti-PE40 blood serum( 1:500 dilution),the second antibody was Pig-anti-Mus IgG(1:1000 dilution) and Benzidine (DAB) was substrate. On the 68 kDa place ,there was an apparent protein blotting band. This indicated that the purpose protein had antigenicity of Cysticercus. because of expression vector pET28a(+) which contain His-Tag in N tip of fusion protein,we could purify protein by Ni²⁺ affinity column. After purified,there was an apparent protein band on 68×10³ Da place and this result was same as fusion protein of pET28-ScFv-PE40. SDS-PAGE indicated that the putity was above 95%, coefficient of recycling of solubility protein was 50%, recycling dose was 5mg / L.To detect cytotoxicity of purpose protein,experiment and kill and wound arcuation was made. The arcuation indicated that the numbers of morpho-complete oncosphere of handle group and control group were showed fall-off tendency. But the fall-off tendency of arcuation with the group which contained recombinant toxinum was obviously more swift than control group(P<0.05).Compared with three arcuations,it was indicatied that there was strong killed action for recombinant toxinum to oncosphere and the effect of recombinant toxinum had dose dependence.