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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2022, Vol. 48 Issue (2): 247-253    DOI: 10.3785/j.issn.1008-9209.2021.03.251
动物科学与动物医学     
捻转血矛线虫酵母双杂交cDNA文库的构建及鉴定
张惠(),黄艳(),周静茹,吴飞,童丹妮,陈学秋,杨怡,马光旭,杜爱芳()
浙江大学动物科学学院动物预防医学研究所，浙江省动物预防医学重点实验室，杭州 310058
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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摘要：

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    
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 words: Haemonchus contortus    cDNA library    yeast two-hybrid system    protein-protein interaction
收稿日期: 2021-03-25 出版日期: 2022-04-29
CLC:  S 855.9  
基金资助: 国家自然科学基金(32002304);国家重点研发计划(2017YFD0501203);浙江省基础公益研究计划(LGN20C180005)
通讯作者: 杜爱芳     E-mail: 21817048@zju.edu.cn;afdu@zju.edu.cn
作者简介: 张惠（https://orcid.org/0000-0002-0178-8915），E-mail：21817048@zju.edu.cn|张惠（https://orcid.org/0000-0002-0178-8915），E-mail：21817048@zju.edu.cn
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张惠
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周静茹
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陈学秋
杨怡
马光旭
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引用本文:

张惠,黄艳,周静茹,吴飞,童丹妮,陈学秋,杨怡,马光旭,杜爱芳. 捻转血矛线虫酵母双杂交cDNA文库的构建及鉴定[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(2): 247-253.

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.

链接本文:

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

图1  捻转血矛线虫的总RNAM：DL10000 DNA分子标志物；1～7：总RNA样品。
  
图3  cDNA均一化效率检测M：DL2000 DNA分子标志物；1：N10C；2：N16C；3：N22C； 4：N28C；5：UN10C；6：UN16C；7：UN22C；8：UN28C（N：均一化模板；UN：未均一化模板；C：循环数）。
图4  cDNA文库滴度鉴定
图5  cDNA文库重组率和插入片段cDNA长度M：DL2000 DNA分子标志物；1～24：插入片段的PCR产物。
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