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Received: 2015-01-08

Revision Accepted: 2015-07-06

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Hong-cui Liu

http://orcid.org/0000-0002-3531-6246

Shao-nan Li

http://orcid.org/0000-0001-8158-7891

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Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.2 P.110-126

http://doi.org/10.1631/jzus.B1500008


Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna


Author(s):  Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li

Affiliation(s):  Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China; more

Corresponding email(s):   snli@zju.edu.cn

Key Words:  Daphnia magna, Cholinesterase (ChE), Polymerase chain reaction (PCR), Recombinant protein ChE, Enzyme-linked immunosorbent assay (ELISA), Triazophos


Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna[J]. Journal of Zhejiang University Science B, 2016, 17(2): 110-126.

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%T Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
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T1 - Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
A1 - Hong-cui Liu
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EP - 126
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PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1500008


Abstract: 
To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

大型溞胆碱酯酶蛋白的原核表达及其多克隆抗体的制备和适用性研究

目的:原核表达并纯化大型溞胆碱酯酶(ChE),制备鼠抗大型溞ChE多克隆抗体,并对抗体的适用性进行研究。
创新点:首次通过原核表达获得了大型溞重组ChE蛋白,通过免疫小鼠获得了高效价、高特异性的多克隆抗体,通过样品检测证明了抗体的适用性。
方法:利用聚合酶链式反应(PCR)技术获得大型溞ChE基因编码序列,并将其亚克隆至原核表达载体pET-29a(+),构建重组表达质粒pET-ChE,用异丙基硫代半乳糖苷(IPTG)诱导重组蛋白的表达;对表达产物进行聚丙烯酰胺凝胶电泳(SDS-PAGE)和酶活性检测,并对蛋白进行纯化(图4);使用纯化蛋白免疫BALB/c小鼠,制备多克隆抗体(图5),用酶联免疫吸附分析法(ELISA)对处于三唑磷和低温胁迫下以及经反复冻融处理的大型溞体内的ChE含量变化进行检测(图7、表6和7),以评价抗体的可适用性。
结论:获得了高纯度的ChE重组蛋白;免疫小鼠后,获得了特异性强、高效价的抗体;通过对处于三唑磷和低温胁迫下以及经过反复冻融处理的大型溞体内的ChE含量的检测,证明了抗体的适用性。

关键词:大型溞;胆碱酯酶;聚合酶链式反应(PCR);重组胆碱酯酶蛋白;酶联免疫吸附分析法(ELISA)

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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