Construction And Expression Of Engineering Antibody Fragments Chimeric Fab And Chimeric F(ab')₂ Against Human Hepatocarcinoma

Aim:High level expression of purpose gene is an important precondit- ion for therapeutic antibody with application value. Now small molecular antibodies have become popular because of its wide uses in diagnostic and therapeutic function in addition to research use. To improve protein yields and get high affinity and specific anti-HAb18G antibody, we efficiently expressed it and get chimeric Fab and chimeric F(ab')2 antibody against human Hepatocarcinoma in Ecoli, and we also chose to express chimeric Fab it in Pichia pastoris in order to get high level expression, The expressed antibody has been analyzed by SDS-PAGE, Western-blot and ELISA ,and laid a solid foundation for its further application.Methods: All the studies can be divided into five parts according to experiments'aims. The protocols were briefly described as the following: 1. Expression as inclusion bodies in E.coli and correctly folding of chimericF(ab')2 antibody against human Hepatocarcinoma .The cFd and cL chain genes were amplified by PCR with specific primers containing relative restriction endonuclease sites and with pET32a/cFab as template.Two cFd and cL fragment tegethered by two peptide linkers:Linker32and Linker21,got the two-pair fragments: â‘  cL-Linker32-cL and cFd-Linker32-cFd,OcL-Linker21-cL and cFd-Linker21-cFd. After digestion by restriction endonuclease, cL cloned genes were inserted into prokaryotic expression vector pET21a , cFd cloned genes into vector pET32a , constructed four non-fusion prokaryotic expression vectors: pET32a/cFd2-32 and pET21a/cL2-32, pET32a/cFd2-21 and pET21a/cL2-21. Then, recombinant vectors were transformed to E.coli JM109(DE3) and efficiently expressed through induced by IPTG. Whether high efficient expression was fulfilled can be checked by SDS-PAGE electrophoresis analysis. Moreover, a large amount of cFd₂-32 and cL₂-32,, cFd2-21 and cL₂-21 products were prepared. And then, they were mixed with equal Molar of quantity to refold into cF(ab')2 by gradient dialysis. The refolded product was identified and analyzed by SDS-PAGE electrophoresis , Western-blot assay and ELISA.2. High secretory expression of the chimeric Fab against human Hepatocarcinoma in E. coli and bioactivity determinat.The cFd and cL genes were amplified by PCR with specific primers. PCR products were digested and orderly inserted into vector PYZ to construct an expression vector PYZ/cFab. After recombinant vector was transformed into is.coli 16C9, soluble expression induced by low phosphate culture medium . Moreover, the location of expression products were determinated by ELISA. At last, interested protein was purified by protein G affinity column, and purified product was detecteded by SDS-PAGE electrophoresis, western-blot assay, ELISA and immunofluorescense staining to confirm its molecular weight and immunological activity.3. Construction of engineering chimeric F Cab') 2 antibody against human hepatoma and high secretory expression in E.coli.The plasmid expressing antibody fragment cF (ab')2 was obtained through remaking the DNA sequence of Fab. Imitating the hinge domain structure included two sequences: (CPP) 3 and ( TC) 2, the C-term of a DNA sequence which coding the domain CH1 of H-chain was amplified and decorated with thePCR technology. The former DNA sequence was replaced by two new modified sequences and two expression vectors of F (ab')2 was formed.This plasmid was transferred into E coli 16C9 and the solutable fragments F (ab')2 were secreted into the periplasmic space. After affinity purification using protein G, the fragments F(ab') 2 were gained. The productions were detecteded by SDS-PAGE electrophoresis and western-blot assay and ELISA and immunofluorescense staining to confirm its molecular weight and immunolo-gical activity.4. High secretory expression of the chimeric Fab against human Hepatocarcinoma in Pichia pastoris.Genes of cL chain and cFd fragment of cFab antibody from the plasmids pET32a/ Fab were subcloned into vectors pPIC9K and pPICZaA respectively, whose sizes were about 700bp. The genes contain right primer sequence and restrictive endonuclease sites. Firstly the recombinant plasmids pPIC9K/cL was transformed into Pichia pastoris strain GS115 by electroporation and the transfonnants were inoculated to the YPD plate with G418 and the multiple inserts clones was obtained. Patch the transfonnants on both an MM plate and an MD plate, the results showed transfonnants phenotype were Mut+. The clone of pPIC9K/L transformant was induced by methanol and the optimized expression condition is 1% methanol concentration and pH 6.0 medium. The induced product was analyzed by SDS-PAGE and Western blotting,.The linearized recombinant plasmids pPICZaAcFd is transducted into the genome of GS115 Pichia pastoris which genome has cL chain . Mutiple insert transfonnants were screened by G418 and Zeocin. The optimized expression condition is 1% methanol concentration and pH 6.0 medium. The production was detected by SDS-PAGE , ELISA and Western blotting assay after purification. Results:1. Two-pairs of prokaryotic expression vectors pET21a/cL2 — 32 and pET32a/cFd2-32, pET21a/cL2-21 and pET32a/cFd2-21, were successfullyconstructed ,and four fragments were high efficiently expressed by IPTG induction in E.coli JM109 (DE 3) , The expression products exists mainly in the form of inclusion body, and the relative molecular weight of which is proximately 50 KD or so, and whose quantity was about 26.1%, 24.6%, 30.1%, 36.2% of total bacterial proteins, respectively. In addition, under the condition of denaturation and renaturation, Products were analyzed by SDS-PAGE electrophoresis, the results indicated that Fd2 32 and cL2-32 chain were successfully refolded together into cFab by means of gradient dialysis, and the relative molecular weight was 90KD or so with about 14% of refolding recovery, the renaturation efficiency of the Fd2 21 and cL2-21 chain too low ,only about 5% . Western-Blot assay and ELISA results showed that renatured protein could more specifically bind to HAb18G than . 2.The cFd and cL genes of cFab were successfully amplified, a part of phoA and STII genes linked with them,respectively, whose sizes were 810bp and 693bp.All genes contain right primer sequence and restrictive endonuclease sites.The results of restrictive enzyme digestion show that the expression vector PYZ/cFab has been constructed successfully. After recombinant vector was transformed into E.coli 16c9, induced by low phosphate culture medium, and soluble expression products mainly existed in periplasmic space of E.coli. The results of SDS-PAGE electrophoresis and Western-Blot assay showed that the molecular weight of intereted protein was approximately 45 KD under unreduction conditioned 25KD after treatment with reduction reagent, and that all bands can bind with goat anti-human IgG.3. The method of construction and identification as the former. The nucleotide sequences of cFd- (CPP) 3 and cFd- (TCPPCPA) 2 were right proved by sequencing. This plasmids were transferred into the E coli 16C9 and the solutable fragment F (ab') 2 were secreted into the periplasmic space. After affinity purification using protein G , the fragment F(ab')2 was gained.the products were analyzed by SDS-PAGE under reducing conditions, which showed that the molecular weight of intereted protein was approximately 90KD under unreduction condition,and 45KD after treatment with reduction reagent, and that all bands can bind with goat anti-human IgG. The concentration of the cF(ab')2 with (CPP) 3 was 810μg/L examined by Gel-Protein software. The result of ELASI demonstrated that F(ab' ) 2 could more specifically bind to HAbl8G than cFab. Immunofluorescense staining to confirm its immunological activity.4. Genes of cL and cFd fragment from the plasmids pET32a/ cFab were subcloned into vectors pPIC9K and pPICZaA respectively, whose sizes were about 700bp. The genes contain right primer sequence and restrictive endonuclease sites. The analysis results of recombinant vectors by restriction endonuclease digestion showed that cL chain and cFd fragment genes were correctly inserted into pPIC9K and pPICZaA.The results of sequencing analysis showed that cL chain and cFd fragment genes were completely in coincidence with the sequences known and all of them have the right opening coden reading frame. The recombinant plasmids pPIC9K/cL was transformed into Pichia pastoris strain GS 115 by electroporation and the transfonnants were inoculated to the YPD plate with G418 and the multiple inserts clones was obtained. A clear band of 660bp was seen in the PCR product with genomic DNA of the pPIC9K/cL clone. Transformed with pPIC9K clone was used as control and without the band. This confirmed that cL gene had been integrated into the yeast chromosomal DNA. Patch the transformants on both an MM plate and an MD plate, the results showed transformants phenotype were Mut+. The clone of p?IC9K/cL transformant was induced by methanol and the optimized expression condition is 1% methanol concentration and pH 6.0 medium. The induced product was analyzed by SDS-PAGE and Western blotting, which showed a new band of 23 KD and suggested that cL chain was expressed successfully. The linearized recombinant plasmids pPICZaAFd is transducted into the genome of GS115 Pichia pastoris which genome has cL chain . Mutiple insert transformants were screened by G418 and Zeocin and transformant DNA is extracted and detected by PCR using the cFd primers. A...