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Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (3): 317-324    DOI: 10.3785/j.issn.1008-9209.2018.09.281
Food sciences     
Secretory expression of recombinant human acetylcholinesterase gene in Pichia pastoris and evaluation of its sensitivity to pesticides
Fangfang YANG1(),Jiahui LI1,Sainan ZHANG1,Zhen WANG2,Zhiyin LIAO1,Shoufeng WANG1,3()
1. Department of Basic Medicine, Zhejiang University, Hangzhou 310058, China
2. Greentown Agricultural Testing Technology Ltd. , Hangzhou 310052, China
3. Zhejiang Provincial Key Laboratory of Microbial Biochemistry and Metabolism Engineering, Hangzhou 310058, China
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Abstract  

Acetylcholinesterase (AChE) is a key enzyme for the detection of organophosphorus and carbamate pesticide residues by enzyme inhibition method. In this study, human acetylcholinesterase (hAChE) was secreted and expressed by Pichia pastoris expression system, and its sensitivity to eight pesticides was evaluated, which laid the foundation for large-scale industrial production of highly sensitive enzyme sources. Primers F1 and R1 were designed according to the published gene sequence of hAChE in GenBank. Then the hAChE gene coding region sequence was amplified by high fidelity PCR using pReceiver-M02-hAChE-ORF plasmid as the template, and the recombinant expression vector pPIC9K-hAChE was constructed by inserting into the vector pPIC9K through double enzyme digestion of SnaB I and Avr Ⅱ. The transformant was selected to sequence and verify the accuracy of the sequence. The linearized pPIC9K-hAChE by Sal I was electro-transformed into P. pastoris GS115. The recombinant strains were screened by minimal dextrase medium (MD) plate and G418 resistance gradient plate. Finally, the positive strain colony was screened on the yeast extract peptone dextrose medium (YPD) plate with 4.00 mg/mL G418 resistance and was identified by polymerase chain reaction (PCR). The expression of the recombinant strain was induced by 0.5% methanol for 144 h. The sodium dodecyl sulfonate (SDS)-polyacrylamide gel electrophoresis map of the fermentation supernatant showed an obvious protein band at 65 kDa, and the size was consistent with the expected hAChE protein. The purified hAChE was obtained using a Q-Sepharose FF chromatography. The content of hAChE protein in the fermentation supernatant was 1.330 mg/mL and accounted for 5.58% in total protein by Bradford method. The highest enzyme activity reached 1 080 nmol/(min?mL) after purification. By preliminary calculation of the 50% inhibitory concentration (IC50) of the eight common pesticides of the fermentation supernatant, it was found that trichlorfon>dichlorvos>arbofuran>malathion>dimethoate>Methamidophos>Carbaryl>chlorpyrifos, suggesting that hAChE is most sensitive to chlorpyrifos.



Key wordshuman acetylcholinesterase      Pichia pastoris      secretory expression      protein purification      enzyme activity determination      pesticide sensitivity     
Received: 28 September 2018      Published: 25 June 2019
CLC:  Q 78  
  Q 814  
Corresponding Authors: Shoufeng WANG     E-mail: 2415090314@qq.com;sfwang@zju.edu.cn
Cite this article:

Fangfang YANG,Jiahui LI,Sainan ZHANG,Zhen WANG,Zhiyin LIAO,Shoufeng WANG. Secretory expression of recombinant human acetylcholinesterase gene in Pichia pastoris and evaluation of its sensitivity to pesticides. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 317-324.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2018.09.281     OR     http://www.zjujournals.com/agr/Y2019/V45/I3/317


重组人乙酰胆碱酯酶基因在毕赤酵母中分泌表达及其对农药的敏感性评估

乙酰胆碱酯酶是用酶抑制法检测有机磷和氨基甲酸酯类农药残留的关键用酶,本研究利用毕赤酵母表达系统分泌表达人乙酰胆碱酯酶,并评估其对8种农药的敏感性,为工业化大规模生产高敏感性酶源奠定基础。根据GenBank上已发表的人乙酰胆碱酯酶(human acetylcholinesterase, hAChE)基因核酸序列设计引物,从pReceiver-M02-hAChE-ORF质粒中扩增出hAChE的开放阅读框(open reading frame, ORF)片段,接着利用SnaB Ⅰ和Avr Ⅱ双酶切将其克隆至载体pPIC9K中,构建pPIC9K-hAChE表达载体,然后将Sal Ⅰ线性化的pPIC9K-hAChE电转化至毕赤酵母GS115中,在含4.00 mg/mL G418抗性平板上筛选获得阳性克隆,最后经0.5%甲醇诱导分泌表达144 h,发现其发酵上清液的十二烷基磺酸钠-聚丙烯酰胺凝胶电泳图谱在65 kDa处有一条明显的蛋白条带,大小与预期的hAChE蛋白一致。利用Q-Sepharose FF柱层析法对其总蛋白进行纯化,获得了较纯的人乙酰胆碱酯酶。用Bradford法测得发酵上清液中hAChE蛋白质量浓度为1.330 mg/mL,占总蛋白的5.58%,纯化后比酶活达到1 080 nmol/(min?mL)。通过初步计算比对重组工程菌发酵上清液对8种常见农药的半抑制浓度,得出敌百虫>敌敌畏>克百威>马拉硫磷>乐果>甲胺磷>甲萘威>毒死蜱,即人乙酰胆碱酯酶对毒死蜱最敏感。


关键词: 人乙酰胆碱酯酶,  毕赤酵母,  分泌表达,  蛋白纯化,  酶活测定,  农药敏感性 

引物名称

Primer name

引物序列( 5 ' 3 '

Primer sequence ( 5 ' 3 ' )

hAChE F1 AGCTACGTATATGAGGCCCCCGCAGTG
hAChE R1 TACCCTAGGATCACAGGTCTGAGCAGC

通用引物α-因子

Universal primer α-factor

TACTATTGCCAGCATTGCTGC

通用引物 3 ' AOX

Universal primer 3 ' AOX

GCAAATGGCATTCTGACATCC
Table 1 Primer sequences for hAChE gene cloning and colony PCR analysis
Fig. 1 Amplified PCR product of hAChE gene
Fig. 2 PCR identification result of recombinant expression vector pPIC9K-hAChE positive colony
Fig. 3 Screening results of G418 resistant plates of recombinant yeast GS115/pPIC9K-hAChE
Fig. 4 PCR identification result of positive colony of recombinant hAChE resistant yeast strain
Fig. 5 SDS-PAGE analysis of fermentation supernatant protein
Fig. 6 Enzyme activity-time curve of fermentation supernatant
Fig. 7 SDS-PAGE analysis of purified target protein
Fig. 8 Comparison of inhibitory rates of eight common pesticides on hAChE
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