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Journal of Zhejiang University (Agriculture and Life Sciences)  2022, Vol. 48 Issue (2): 247-253    DOI: 10.3785/j.issn.1008-9209.2021.03.251
Animal sciences & veterinary medicines     
Construction and identification of yeast two-hybrid cDNA library of Haemonchus contortus
Hui ZHANG(),Yan HUANG(),Jingru ZHOU,Fei WU,Danni TONG,Xueqiu CHEN,Yi YANG,Guangxu MA,Aifang DU()
Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
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Abstract  

This experiment aimed to construct a cDNA library of Haemonchus contortus, which could provide the basis for further research on the protein interaction mechanism and screening out the interactive proteins of H. contortus. The L3 stage larvae of H. contortus were used to extract the total RNA by the TriZol method. Besides, the kit was adopted to construct a cDNA library of H. contortus and the cDNA was normalized. Then, the purified cDNA were transformed into yeast Y187 cells together with the linear pGADT7-Rec, and a yeast two-hybrid cDNA library of H. contortus was constructed by homologous recombination. The results showed that a homogenized yeast activation domain (AD) library with the recombinant rate of 100%, average inserted fragment length of 1 000 bp, and working fluid cell density of more than 3.5×107 CFU/mL was constructed. The constructed expression library met the requirements of yeast two-hybrid screening. This library lays a foundation for the molecular mechanism study and vaccine development of H. contortus.



Key wordsHaemonchus contortus      cDNA library      yeast two-hybrid system      protein-protein interaction     
Received: 25 March 2021      Published: 29 April 2022
CLC:  S 855.9  
Corresponding Authors: Aifang DU     E-mail: 21817048@zju.edu.cn;afdu@zju.edu.cn
Cite this article:

Hui ZHANG,Yan HUANG,Jingru ZHOU,Fei WU,Danni TONG,Xueqiu CHEN,Yi YANG,Guangxu MA,Aifang DU. Construction and identification of yeast two-hybrid cDNA library of Haemonchus contortus. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(2): 247-253.

URL:

https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2021.03.251     OR     https://www.zjujournals.com/agr/Y2022/V48/I2/247


捻转血矛线虫酵母双杂交cDNA文库的构建及鉴定

为构建捻转血矛线虫cDNA文库,进一步研究捻转血矛线虫蛋白互作机制以及为筛选捻转血矛线虫互作蛋白提供依据,本研究以捻转血矛线虫L3期幼虫为材料,利用TriZol法提取捻转血矛线虫总RNA;使用试剂盒构建捻转血矛线虫的cDNA文库,并对cDNA进行均一化处理,再将纯化后的cDNA与线性化的pGADT7-Rec重组构建的文库质粒转化至Y187酵母中,构建出酵母转录激活结构域(activation domain, AD)文库。结果表明:本研究构建了重组率为100%,插入片段平均长度为1 000 bp,工作液细胞密度大于3.5×107 CFU/mL的捻转血矛线虫均一化酵母AD文库;所构建的表达文库各项指标均达标,符合酵母双杂交筛选要求。本文库为捻转血矛线虫的分子机制研究以及疫苗的开发奠定了基础。


关键词: 捻转血矛线虫,  cDNA文库,  酵母双杂交系统,  蛋白质间相互作用 
Fig. 1 Total RNA extracted from H. contortusM: DL10000 DNA marker; 1-7: Total RNA samples.
 
Fig. 3 Efficiency test of cDNA normalizationM: DL2000 DNA marker; 1: N10C; 2: N16C; 3: N22C; 4: N28C; 5: UN10C; 6: UN16C; 7: UN22C; 8: UN28C (N: Normalized template; UN: Unnormalized template; C: Cycle number).
Fig. 4 Test of cDNA library titer
Fig. 5 Recombinant rate of cDNA library and the inserted cDNA lengthM: DL2000 DNA marker; 1-24: Inserted PCR fragment products.
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