| Diethylstilbestrol is the first artificially-synthesized orally-taken active nonsteroidal estrogen. While DES residue was found in animal origin foodstuff such as liver, muscle, eggs, and milk, and it was accumulated in soil and water over a long period, then the vicious circle of environmental pollution will be induced at last. The pollution may lead to the possibility of canceration in human being and animals and the hazard of child precosity and masculine feminization by the cycle of food chain. International Agency for Research on Cancer has recognized DES as a definite carcinogen. Therefore, the development of sensitive and rapid detection methods for determinating DES residue is essential. The immunological detection is economic, simple and rapid. Therefore, this way of detection is based on the DES-specific antibody. Single chain variable fragment (scFv) which is the engineering antibody has been widely used in medical researches because of the characters such as minimal immunogenicity, small molecule, high tissue penetration, low-cost and consistent production. Currently, scFv fragments are often displayed at the surface of ribosomes or phage, and be made by ribosome display or phage display technology. Naive libraries are generated from genetic materials that have never been exposed to the antigen(s) prior to library construction. Therefore, as long as the library is sufficiently large, the antibodies against any antigen will be obtained from naive libraries. Although recombinant antibody technology has allowed us to isolate antibodies against any antigen, there have been few reports on selecting scFvs against haptens from a naive library by ribosome display technology. Currently, There have been no reports about selection of scFvs against DES by ribosome display.We applied a ribosome display technique to a mouse naive scFv library to select single chain variable fragments (scFvs) specific for DES. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody fragments from a naive library. The main results are as follows:1. The mRNA of spleen cell from an about 6-8 weeks old Balb/c mouse without immunization was extracted for construction antibody library. Single cDNA was synthesised by oliger(dT)15 primers. The sizes of amplificated VH and VL were about 381 bp and 349 bp by RT-PCR, respectively. A 81bp DNA linker containing a sequence encoding (Gly4Ser)3 was amplified, and the result of sequnceing was in conformity with the sequnce in theory. The VH and VL were ligated with the linker (Gly3Ser)4 using SOE-PCR. The assembled scFvs were of expected approximately 750bp size.80% individual clones were correctly in reading frame and no stop codon. The amine acid sequences of the CDRs of scFvs were different. It showed that the constructed library was diversity and could be used to selection of specific scFvs successfully.2. We have amplified T fragment (T7 promoter,3’loop, ribosome binding site) and spacer sequence P fragment (spacer,5’loop) from pT7PD vector. The two amplied fragments were sequencing, and the results of sequnceing were in conformity with the sequnces in theory. The scFv library DNA was placed in the context of the E.coli ribosome-display format by splice-overlap extension and PCR. The assembled 1.2 kb scFv DNA contained a T7 promoter and ribosome binding site and a spacer protein.3. Quality evaluation of a naive library(1) The whole assembled library were subcloned into a clone plasmid vector and following transformation. Nucleotide sequence data indicated that 9 sequences for in vitro transcription and translation were incorporated in frame with the scFv sequences and the phage protein D spacer sequence. In addition, no stop codon was detected in any of the reading frame.Then,the whole library were prepared for the transcription and translation in vitro.(2) The result suggested the library was able to amply effectively and has the abilities of transcription and reverse trascription.4. A mouse naive scFv library was transcripted and translated in vitro to select single chain variable fragments (scFvs) specific for DES. The way of the alterated selection in solution and immobilized in the microtitre wells was used to pan antibody-ribosome-mRNA complexes. The progress of panning was monitored by examining the intensity of RT-PCR products on agarose gel. By the end of the first round, only a weak DNA band was visible.The quantity of RT-PCR products continually increased during the next rounds of panning. Based the above results, enrichment of specific scFvs was clearly confirmed.5. The seventh round scFv library DNA of 100 individual clones were sequenced.43 individual clones of all had no stop codon and were in correct reading-frame, which were cloned into E. coli for soluble scFvs expression.6 The 26 of 43 individual clones selected after the seventh round was inserted into the exprssion vector Pitg-trx.The expression vector was transformed to the expression bacterial BL21(DE3), and supernatants were each extracted to verify the location and functionality of each soluble scFv. The expression of each scFv was confirmed by SDS-PAGE analysis and Western Blotting, and a band at approximately 30KDa was apparent.7. The 26 crude expressed extracts specific to the DES conjugates were assessed for their specificity to free DES by indirect ELISA and a competition assay. Five of the scFv antibodies were specific to DES.8. The coding sequences of the 5 positive scFv clones were identified, and these sequences displayed a high level of homology with each other. Equilibrium dissociation constants (KD) were determined by SPR. The KD for 30-3 was estimated to be 3.79×10-6M. The recombinant scFv was one-step purified by using Ni-NTA affinity agarose chromatography column. |
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