Construction, Expression And Immunological Characteristics Of Three Recombinant Antibodies Against Leucomalchite Green

Malachite Green(MG) is a kind of triphenylmethane dye which was antimicrobial and antiparasitic effectively. It was used as fungicide and repellents in aquatic animal. After absorbed by fish, MG was reduced to be colorless lipophilic metabolite, Leucomalchite Green(LMG). Both MG and LMG are carcinogenic, mutagenic and teratogenic on human health, so they are banned to be used in aquaculture in our country. But it is still being used illegally due to its low cost and efficacy. Therefore, it is important to develop a simple and effective detection method to strengthen supervision. Immunoassay has been proven to be fast, simple and high throughout alternative, which is developed as an effective detection tool in food safety. Antibody is its core material. As the third generation of antibody, engineering antibody, it has advantages of low cost and short time of production, which has been the hot research of many researchers in many countries.In the previous study, anti-LMG single chain antibody(LMG-sc Fv) has been obtained. LMG-Fab and LMG-sc Fab were constructed though the alteration of LMG-sc Fv. Ic ELISA based on three recombinant antibodies were developed and Specificity and stability.of three recombinant antibodies were analyzed. The major research contents and the results are as follows:(1) The construction ofthree recombinant plasmidsAccording to the previous report, the primer of the gene of the Bir A enzyme was designed. After the PCR reaction using the E.coli DH5α as template, the gene about 963 bp of the Bir A enzyme was obtained. The sequence of the gene was blast in the NCBI. The result of blast shown that homologous rate was 100%, which proved the gene of Bir A enzyme was cloned successfully. The gene of LMG-sc Fv and the gene of Bir A enzyme were linked into the plasmid p Comb3 xss to construct recombinant plasmid p Comb3xss-LMG-sc Fv-Bir A.VH gene and VL gene were cloned using the recombinant plasmid p Comb3xss-LMG-sc Fv-Bir A as template. CH1 gene and CL gene were cloned using p3 MH as template. The VH gene and The CH1 gene were linked through overlap extend PCR method to construct Fd gene about 663 bp, while the VL gene and the CL gene were linked to construct κ gene about 642 bp. The Fd gene and the κ gene were inserted re Spectively into the plasmid p Combxss to construct the recombinant plasmid p Comb3xss-LMG-Fab.The Fd gene and the κ gene were cloned using p Comb3xss-LMG-Fab as template. They were linked with linker(Gly4Ser)3 usind the overlap extend PCR to construct κ-linker-Fd(sc Fab). The sc Fab gene was inserted into the plasmid p Comb3 xss.(2) Expression of three recombinant antibodiesAfter transformed into E.coli Top10f’, the recombinant E.colis were induced with isopropyl-β-d-thiogalactoside(IPTG). The result of SDS-PAGE and Western Blotting demonstrated that LMG-sc Fv about 28 k Da were expressed successfully, which was labeled with biotin during the process of expression; there is one band about 27 k Da of LMG-Fab which was the κ chain of Fab and there is one band about 54 k Da were LMG-sc Fab.(3) Immunological characteristics of three recombinant antibodiesAfter optimized the reaction conditions, ic ELISA based on three recombinant antibodies were developed. Ic ELISA based on LMG-sc Fv-Biotin, the IC50 was 20.11 ng/m L and the linear range of detection was 4.11 ~ 98.56 ng/m L; based on LMG-Fab, the IC50 was 45.56 ng/m L and the linear range of detection was 12.80 ~ 162.13 ng/m L; based on LMG-Fab, the IC50 was 37.48 ng/m L and the linear range of detection was 10.28 ~ 136.68 ng/m L.The analogues were detected by ic ELISA based on the three recombinant antibodies. Cross reaction studies showed that LMG-sc Fv-Biotin, LMG-Fab and LMG-sc Fab protein did not cross react obvilously with LMG analogs, excepted for Leucocrystal Violet(LCV).The analysis of the stability was that the LMG-Fab was the most stable, LMG-sc Fab secondly and The sc Fv was most unstable.