犬耳螨全长cDNA文库的构建

The Construction of Full-Length cDNA Library for Otodectes cynotis.

作者信息

Hu Li, Zhao Ya-E, Niu Dong-Ling, Yang Rui, Zeng Ji-Hui

机构信息

Department of Pathogenic Biology and Immunology, School of Basic Medical Sciences, Xi'an Jiaotong University, No.76 Yanta West Road, Xi'an, 710061, China.

Xi'an PET Animal Hospital, Xi'an, China.

出版信息

Acta Parasitol. 2019 Jun;64(2):251-256. doi: 10.2478/s11686-019-00034-y. Epub 2019 Mar 12.

DOI:10.2478/s11686-019-00034-y

PMID:30864098

Abstract

BACKGROUND

Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging.

METHODS

To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability.

RESULTS

Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/μl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 10 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively.

CONCLUSION

The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.

摘要

背景

犬耳螨（Otodectes cynotis，赫林，1838年）是一种在全球范围内传播的耳螨病病原体。这种螨虫主要感染肉食动物，有时也会感染人类。然而，由于缺乏cDNA文库，对其发病机制的研究一直具有挑战性。

方法

为了解决这个问题，本研究首先从中国西安一只感染耳螨的猫身上采集了犬耳螨样本用于RNA提取。然后，使用SMART技术构建全长cDNA文库。最后，对从cDNA文库中特异性扩增出的大于500 bp的阳性克隆和Hsc70-5基因片段进行测序和分析，以验证文库的可靠性。

结果

结果显示，从300只螨虫中提取的RNA质量良好，浓度为149 ng/μl，OD260/OD280为1.99。该文库符合优质文库的质量标准，滴度为5.02×10 PFU/ml，重组率为97.61%。此外，克隆4和Hsc70-5与GenBank中犬耳螨的Ef1-α和Hsc70-5基因序列的同一性分别为98.38%和99.72%。