| Heavy metals are a kind of contaminants that can lead to side effects even if they arepresent in extremely minute quantities. They are the most insidious pollutants because oftheir nonbiodegradable nature and ability to persist for long periods. All forms ofecological systems are effected to various extends by heavy ,metals. Through the foodchain, heavy metals may deposit into the humane body, cause serious health problems,specifically cancer, and even transfer to the next generation on the genetic level. Therefore,the quantitative analysis of trace heavy metals is extremely important in environmental andagricultural food monitoring,To establish hybridoma cell lines that can secrete anti-Cd/Pb monoclonal antibody(McAb) with high affinity and specificity, and develop an effective enzyme-linkedimmunosorbent assay(ELISA) for Cd/Pb. Based on the identification of their affinity,specificity and function, clone the variable region genes of the McAb and constructrecombinant anti-metal single chain Fv (ScFv) gene, then transform and express the ScFvgene in E. coli, and identify the resulting protein. According to variable region gene athree-dimensional model of ScFv of anti-Cadmium single chain antibody was mimiced.Heavy metal was coupled to protein carrier (keyhole limpet hemocyanin, KLH; bovineserum albumin, BSA) via bifuntional chelate reagents (1-(4-isothiocyanaobenzyl)-ethylene-diamine N, N, N', N'-tetraacetic acid; ITCBE; S-2(4-aminobenzyl)-diethylenetriaminepentaccetic acid; 9-NH2-Bn-DTPA; S-2-(4-isothiocyanaobenzyl)-diethylenetriaminepentaccetic acid, SCN-Bn-DTPA). The hybridoma cell lines that can secrete anti-metal (Cd,Pb) McAbs with high affinity were established by hybridoma technique. Purified McAbs were prepared by salt precipitation and protein chromatography. The titer and affinity ofMcAbs were determined by indirect ELISA, the class and subclass of McAbs wereidentified by Monoclonal Antibody Isotyping Kit, the specificity of McAbs was determinedby competitive inhibition ELISA. Under the optimized blocking reagents, ionic strengthand pH value, the standard curves were established by indirect competitive ELISA. Thetotal RNA of hybridoma cell lines was isolated with Trizol reagent, and the variable regiongenes were amplified with variable region primers by RT-PCR. The variable region genesof heavy chain and light chain were strung together by splicing by overlap extension(SOE-PCR) and the ScFv gene was constructed. After cloned into pGEM-T Easy vector,the recombinant ScFv gene was identified by endonuclease digestion, PCR and sequencing.The recombinant expression vector pET-heavy metal-ScFv was constructed byendonuclease digestion and ligation, and then transformed into E. coli BL21 (DE3) strain.After screened on LB plate with Kanamycin and identified by endonuclease digestion andPCR, the positive strain was cultured under IPTG. The inclusion body of the inducedbacteria was collected, denaturalized with urea and renaturalized by dialysis withrenaturalizing solution. The molecular weight and immuno-activities of expressed proteinwere identified by SDS-PAGE and competitive inhibition ELISA respectively OnSWISS-MODEL, with homologous protein-structure-prediction, we constructed andanalyzed molecular models of single chain antibody(Aa). Seven hybridoma strains that cansecrete anti-heavy metal McAbs were established designed Aa4, Aa6, Ac4, Ba2, 4/7, 7/4and 6E11. The concentration of the purified antibodies ranges from 1.58 mg/mL to 5.0mg/mL, with titer of 1:200-1:500 and affinity conatants of 108-1010L/mol. Thecross-reactivities in this ELISA were less than 3％for other metal ions. The standard curveswere obtained by the antibodies with higher affinity. The IC50 values of Aa6 specific tocadmium was 4.19μg/mL, showing the lowest detection limit of 0.313μg/mL, the IC50value of 4/7 achieved for lead was 2.72μM, showing the detection range of with the lowestdetection limit of 0.056μM. Recoveries from the analyte-fortified into tap water andultrapure water were in the range of 80-114％. The variable region genes were amplifiedfrom hybridoma cell lines by RT-PCR, and recombinant ScFv gene was constructed. Thesequencing result shows that the ScFv gene consists of about 750bp, with more than 300bpof heavy chain variable region gene and light chain variable region gene, which werelinked by a 45bp peptide. Then the ScFv gene was cloned into expression vector pET-30a(+) by the direction of VH-linker-VL. The recombinant ScFv gene was expressed successfully in E.coli under the induction of IPTG. The anti-heavy metal ScFv protein wasexpressed mainly in the form of inclusion body and with a molecular weight of about29KD. After denaturalization and renaturalization treatment, the immuno-activity ofanti-heavy metal ScFv protein was verified as it can not competitively inhibit the bindingbetween McAband ScFv with inhibitory rate of about 10.6％. The structure model ofantibody is constructed successfully.Seven of hybridoma cell lines that can secreteanti-heavy metal McAb with high affinity and specificity were established. The variableregion genes of the McAbs were amplified and ScFv gene was constructed. Therecombinant anti-heavy metal ScFv gene was then transformed and successfully expressedin E. coli as a fusion protein witn immuno-activity. The three-dimension of anti-cadmiumsingle chain antibody was provided a method for improving antibody affinity, investigatingantibody structure and analyzing function of light, as well as heavy chain in antibodyactivity. |
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