High Level Expression Of Humanized Anti-keratin Antibody And Its Effect On Cultured Keratinocytes

Numerous investigations have showed that anti-keratin auto antibody (AK auto Ab) is a very important element in human immune system. In some pathological states such as psoriasis and contact dermatitis, a certain serum level of the antibody could inhibit the progression of the diseases, and is beneficial for the recovery from the diseases. Studies both in vitro and in vivo animal models indicated that anti-keratin antibody had therapeutic potential for such disorders. Aim :High level expression of purpose gene is an important precondition for therapeutic antibody with application value. Antibodies have diagnostic and therapeutic value in addition to research use. They are often used in the form of small molecular antibodies, which have the advantages of low immunogenicity, rapid accumulation at the targeted tissue and be clean out quickly. We have got the humanized antibodiy wit high affinity and specific against keratin by phage antibody library technology. To improve protein yields and get high affinity and specific anti-keratin antibody, we chose to express it in E. coli BL21 (DE3) and get human anti-keratin Fab by renaturation in vitro, we also chose to express it in Pichia pastoris in order to get high level expression of antibody, which laid a solid foundation for its further application. Methods:All the studies can be divided into three parts according toexperiments' aims. The protocols were briefly described as the following:1. Expression as inclusion bodies in E. coli and folding of human Fab antibody against keratin .The Fd and light chain genes were PCR amplified by specific primers containing relative restriction endonuclease sites with p3MH/Fab as template. After digestion by restriction endonuclease, cloned genes were inserted into prokaryotic expression vector pET32a to construct two non-fusion prokaryotic expression vectors, pET32a/Fd and pET32a/L Then, recombinant vectors were transformed to E. coli BL21(DE3) and induced by IPTG. Whether high efficient expression was fulfilled can be checked by SDS-PAGE electrophoresis analysis. Moreover, a large amount of light and Fd expression products were prepared. And then, they were mixed with equal Molar of quantity to refold into Fab by gradient dialysis. The refolded product was identified and analyzed by SDS-PAGE electrophoresis > Western-blot assay and ELISA.2. Secretory expression of human ScFv against keratin in Pichia pastoris.Subcloning the gene of ScFv of anti-keratin antibody into vector pPIC9K from the plasmids p3MH/ScFv, after confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/ScFv was linearized and transducted into the genome of GS115 Pichia pastoris. After screening of mutiple inserts by G418, we demonstrated desired gene had integrated into the yeast genome by genomic PCR . The mutiple inserts transformants were induced to express secretively by 5 ml/L methanol after phenotypic analysis. The product was identified and analyzed by SDS-PAGE electrophoresis, Western-blot assay and ELISA.3.Secretory expression of humanized Fab of anti-keratin antibody in Pichia pastoris.Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab are subcloned into vectors pPIC9K and pPICZ A respectively, which confirm by DNA sequence analysis. The recombinantplasmids pPIC9KL is transducted into the genome of GS115 Pichia pastoris after linearized. Mut+ mutiple insert transformants are screened by G418 and transformant DNA is extracted and detected by PCR using the L chain primers . After phenotypic analysis, the clone of pPIC9K/ScFv transformant is induced by methanol to express soluble L chain and the expression condition is optimized. Samples of supernatant proteins are analyzed by SDS-PAGE , Western blotting and ELISA assays . The linearized recombinant plasmids pPICZaAFd is transducted into the genome of GS115 Pichia pastoris which genome has L chain . Mutiple insert transformants are screened by G418 and Zeocin and transformant DNA is extracted and detected by PCR using the Fd primers . The e...