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Hong-cui Liu


Shao-nan Li


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


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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author="Hong-cui Liu, Bing-qiang Yuan, Shao-nan Li",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
%A Hong-cui Liu
%A Bing-qiang Yuan
%A Shao-nan Li
%J Journal of Zhejiang University SCIENCE B
%V 17
%N 2
%P 110-126
%@ 1673-1581
%D 2016
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1500008

T1 - Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna
A1 - Hong-cui Liu
A1 - Bing-qiang Yuan
A1 - Shao-nan Li
J0 - Journal of Zhejiang University Science B
VL - 17
IS - 2
SP - 110
EP - 126
%@ 1673-1581
Y1 - 2016
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1500008

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.




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