【作者】 孟宪梅；

【导师】 柳增善；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 河豚毒素(Tetrodotoxin,TTX)是一种常见且危害较大的海洋生物毒素,广泛存在于海洋生物和陆栖等众多生物中,主要见于豚鱼类。豚鱼肉味鲜美,营养丰富,我国和日本等亚洲国家均有食用习惯,中毒事件频频发生,其快速检测方法的研究对预防河豚毒素中毒具有重要意义。本研究成功制备了TTX毒素人工完全抗原TTX-BSA、TTX-OVA;建立了分泌抗TTX毒素单克隆抗体杂交瘤细胞株3D4和4C5,制备并纯化了单克隆抗体(TTX-McAb),对抗体效价、亚类、分子量、亲和力、敏感性、特异性等进行了测定;建立了以单克隆抗体为基础的间接竞争ELISA、胶体金免疫层析试纸条免疫学检测方法,并组装了ELISA检测试剂盒,其性能指标能满足快速、特异、敏感的食品检测要求。为进一步探索利用融合单链抗体的海洋生物毒素高通量筛检方法,对TTX和另外2种重要的海洋贝毒素石房蛤毒素(Saxitoxin,STX)和大田软海绵酸(Okadaic acid,OA)融合单链抗体进行了研究。利用已构建的杂交瘤细胞株,RT-PCR方法获得TTX-McAb的VH和VL基因,SOE-PCR方法组装单链抗体基因TTX-ScFv(VH-Linker-VL),构建其原核表达载体TTX-ScFv-22b和真核表达载体TTX-ScFv-PICZα,获得了相应的具有反应活性的表达蛋白;将3种海洋生物毒素TTX、OA、STX单链抗体基因进行串联,构建了融合基因表达载体OST-ScFv-22b,获得了相应抗3种毒素的融合单链抗体蛋白,经ELISA检测,与3种毒素反应活性较弱。如何提高融合单链抗体反应活性使其能够用于3种毒素的同步筛检方法,有待于进一步研究。更多还原

【Abstract】 Because okadaic acid (OA), saxitoxin (STX) and tetrodotoxin (TTX) are highly harmful to people, they are concernful marine toxins to cause outstanding safty problems of food and environment. It is important to research rapid detection methods of these toxins. In this study, the immunological methods of detecting TTX including a indirect competitive enzyme-linke immunosorbent assay (icELISA) and colloidal gold immune test paper were established, and then, fusion single chain antibody which was used to decteting OA, STX and TTX was developed. This would open up a new way for detecting marine toxins and other food pollutants .Man-made complete antigens (TTX-OVA and TTX-BSA) were successfully prepared by methanal couple, and the conjugation ratios of TTX to carrier proteins BSA or OVA respectively were 14:1 and 6:1. BALB/c mice immuned by complete antigen(TTX-OVA)made immune response to produce antibodies against TTX. The hybridoma cells (3D4 and 4C5) which secreted monoclonal antibody (McAb) against TTX were established by monoclonal antibody technique. The characteristics of TTX-McAb were determined, such as titer, subtype, molecular weight, affinity, sensitivity and specificity. Two hybridoma cell lines were identified that secretes IgG1 monoclonal antibody against TTX, designated 3D4 and 4C5 after subcloned for 3 cycles by indirect ELISA. The average number of the hybridoma chromosomes were 50±2 and 50±4 and exceeded parent cells respectively. ELISA result showed that the titer of the cell culture supernatant were 1:640 and 1:320, antibody titer 5.12×105 and 3.2×105, and the affinity constant 6.2×106 L/mol and 5.9×106 L/mol respectively.The TTX-McAb of 3D4 was manufactured by means of intraperitoneally injecting BALB/c mice and purified by CA-AS method. The result showed that purification ratio was up to 61.24 %, the recovery was 23.48%, and the purity of IgG was up to 90.99%. Experiments of specificity detection to STX, OA, MC and NOD by indirect ELISA was carried out, showing that TTX-McAb performed no cross reaction with OA, MC and NOD except STX. Preparation of TTX-McAb had successfully established to facilitate immunological detection and preparation of single-chain antibody.An icELISA based on TTX-McAb was set up, detecting condition were optimized, TTX standard and mock samples were detected. Optimized ELISA reaction condition as follow: antigen covering concentration was 1.0μg/mL, antibody dilute strength 1:40 000,covering condition to stay overnight at 4℃, competition action at 37 OC for 30 min, action time of antibody enzyme marked 30 min, action time of substrate 10 min. Equation of linear regression was y=25.681x-4.2725, and coefficient correlation R2=0.989, concentration limit 0.05 ng/mL, linear range 0.05～50 ng/mL. TTX standard and mock samples of 216 portions, including crab, shellfish as well as spiral vagina muscle and egg tissue, were detected. Results showed that average recovery were respectively TTX standard samples 88.89%, crab spawn 87.05%, crab muscle 80.96%, snail 83.26%, shellfish 85.59%. Inner and interassay coefficient of variation were smaller than 7% and 10% respectively. ELISA kit of within plate was packaged, and its performance index reached to the fast,specific and sensitive detection requirement. Specificity detection to STX, OA, MC and NOD by indirect ELISA was done, and results indicated that the monoclonal antibody had no cross reaction with OA, MC and NOD except STX.Colloidal gold immune test paper based on monoclonal antibody was developed, and TTX standard samples were detected. Colloidal gold particles of 20 nm were prepared using trisodium citrate as reducer, and colloidal gold probe was prepared, and condition of paper test paper slip was optimized. Slip of test paper was packaged. Results showed that the best coating density for antibody and colloid gold was 8.0μg/mL, the best pH value of coating the colloid gold was 8.5, the lowest detection concentration limit 50 ng/mL. Specificity detection with STX, OA, MC, NOD by indirect ELISA was done, and result indicated that the monoclonal antibody had no cross reaction with OA, Mc and NOD other than STX. The time for guarantee the quality was 8 weeks at 4℃.VH and VL genes of TTX-McAb were amplified from the RNA of hybridoma cell by RT-PCR using VL and VH genes primers designed according to the monoclonal antibody of mouse source, and were ligated with linkers. The single chain antibody gene TTX-ScFv (VH -Linker-VL) was constructed by SOE-PCR. TTX-ScFv gene was 744 bp including VH of 366 bp, VL of 333 bp and linker of 45 bp. Result by NCBI blast analysis showed that VLand VH genes not only consisted with variable region characteristic of the monoclonal antibody of mouse, and but also had high homology with many single-chain antibody registered.Prokaryotic expression vector TTX-ScFv-22b and eukaryotic expression vector TTX-ScFv-PICZαwere constructed and expressed proteins with reactive activity with TTX were obtained. Recombinant plasmid TTX-ScFv-22b was transferred into E.coli BL21(DE3)and gained high performance expression. Molecular wieght of expressed protein was 26.045 ku. Fusion proteins were induced to express by IPTG at 37℃for 6 h with amounting to 51.38% in inclusion, but amounting to 42.14% at 25℃for 12 h in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing to and ultrafiltrating. Activity was analysed by indirect competition ELISA(ic ELISA).TTX-ScFv gene was cloned into expression vector pPICZαA, and recombinant expression vector TTX-ScFv-PICZαwas constructed. After linearrization, recombinant plasmid was transferred into GS115 of Pichia pastoris, and then were induced to express by methanol at 30℃for 72h with amounting to 15.10%. Expressed fusion protein were concentrated and purified by dialyzing and ultrafiltration, and determined by SDS-PAGE. Activity was analysed by icELISA. ELISA results indicated that expressed fusion proteins in prokaryotic and eukaryotic systems performed reactivity with TTX antigens, and activity of the eukaryotic expressed protein was a little higher than the prokaryotic, but both of them were lower significantly than TTX-McAb.At the same time, three ScFv genes, including OA-ScFv, STX-ScFv and TTX-ScFv, were linked to OA-STX-TTX-ScFv by SOE-PCR. OA-STX-TTX-ScFv gene was 2 256 bp, encoding 752 amino acids. Fused single-chain antibody against three toxins were obtained. Molecular weight of expressed protein was about 79.000 ku and consisted with predicted value. Expressed fusion protein induced by IPTG at 37℃for 6 h amounted to 12.70% in inclusion, but expressed fusion protein induced by IPTG at 25℃for 12 h amounted to 10.90% in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing and ultrafiltrating. ELISA results indicated that the expressed fusion protein had weak reactive activity with three toxins respectively. Fusion single-chain antibodies were expressed with bioactivity. How to improve the bioactivity of fusion single-chain antibodies to estiblish broad-spectrum detection methods need to study in the future.更多还原