Amplification of full-length cDNA sequence of <italic style="font-style: italic">Anopheles dirus</italic> vitellogenin gene and expression and purification of its functional domain

Shasha YU; Hui HU; Jie QIN; Jing WANG; Ting LIU; Tingting LIU; Ying WANG

doi:10.16016/j.1000-5404.202012233

您当前的位置：

首页 >

文章列表页 >

Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain

Basic Medicine | 更新时间：2026-01-09

- Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain
- Journal of Third Military Medical University Vol. 43, Issue 12, Pages: 1140-1145(2021)
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)军事预防医学系热带医学教研室
  2.510220 广州，中国人民解放军陆军第七十四集团军医院重症医学科
- 作者简介：
- 基金信息：
  
  the Natural Science Foundation of Chongqing(cstc2018jcyjAX0182);the National Natural Science Foundation of China(81702035);the National Natural Science Foundation of China(81971971)
- DOI：10.16016/j.1000-5404.202012233
  CLC： Q962;Q963;Q966
- Received：29 December 2020，
  
  Revised：2021-02-02，
  
  Published：30 June 2021
- 稿件说明：
移动端阅览
Shasha YU, Hui HU, Jie QIN, et al. Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain[J]. Journal of Third Military Medical University, 2021, 43(12): 1140-1145.
DOI：

Shasha YU, Hui HU, Jie QIN, et al. Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain[J]. Journal of Third Military Medical University, 2021, 43(12): 1140-1145. DOI： 10.16016/j.1000-5404.202012233.

摘要

目的

对大劣按蚊卵黄蛋白原(

Anopheles dirus

vitellogenin

AdVg)基因cDNA的全长序列进行扩增和分析，并对其功能域蛋白进行表达与纯化。

方法

基于前期克隆的AdVg基因cDNA片段，利用RACE PCR技术，扩增AdVg基因的5'末端及3'末端序列。用Vector NTI软件将测序得到的AdVg cDNA片段、5'末端序列、3'末端序列进行校正，拼接得到完整的AdVg cDNA全长序列。对AdVg全长序列进行功能域预测。利用pet28a(+)构建重组质粒进行AdVg功能域蛋白的表达与纯化。

结果

成功获得AdVg基因全长cDNA序列，包括117 bp的5' UTR序列、192 bp的3' UTR序列和6 186 bp的ORF序列。AdVg蛋白主要包含Vit N、DUF1943及vWD 3个结构域。通过构建重组质粒，成功表达和纯化了AdVg Vit N功能域蛋白。

结论

成功克隆了大劣按蚊卵黄蛋白原基因的全长序列，并获得了重组蛋白。

Abstract

Objective

To amplify and analyze the full-length cDNA of

Anopheles dirus

vitellogenin (AdVg) gene

and express and purify its functional domain.

Methods

Based on the cDNA fragments of AdVg gene cloned earlier

the full-length sequence was obtained through rapid amplification of cDNA ends (RACE). The sequences of AdVg cDNA fragment

5' end and 3' end were corrected and assembled to obtain a complete AdVg cDNA full-length sequence by Vector NTI software. Then the full-length sequence of AdVg was used to predict the functional domain. Finally

recombinant plasmid pet28A (+) was used to express and purify the AdVg functional domain.

Results

The full-length cDNA sequence of the AdVg gene was successfully obtained

including a 117 bp 5' UTR sequence

a 192 bp 3'UTR sequence

and a 6 186 bp open reading frame (ORF) sequence. Meanwhile

the analysis showed that the AdVg protein mainly contained 3 structural domains

including Vit N

DUF1943 and vWD. Then we successfully expressed and purified the functional domain AdVg Vit N by constructing a recombinant plasmid.

Conclusion

The full-length cDNA of AdVg is successfully cloned

and the recombinant protein of its functional domain Vit N is obtained in this study.

关键词

Keywords

references

WHO. World malaria report 2019[EB/OL]. [2019-12-04]. https://www.who.int/publications/i/item/9789241565721.

FERGUSON N M. Challenges and opportunities in controlling mosquito-borne infections[J]. Nature, 2018, 559(7715): 490-497. DOI: 10.1038/s41586-018-0318-5.

RANSON H, LISSENDEN N. Insecticide resistance in African Anopheles mosquitoes: a worsening situation that needs urgent action to maintain malaria control[J]. Trends Parasitol, 2016, 32(3): 187-196. DOI: 10.1016/j.pt.2015.11.010.

DUSFOUR I, VONTAS J, DAVID J P, et al . Management of insecticide res istance in the major Aedes vectors of arboviruses: Advances and challenges[J ] . PLoS Negl Trop Dis, 2019, 13(10): e0007615. DOI: 10.1371/journal.pntd.0007615.

周红宁, 卢勇荣. 云南大劣按蚊生态习性, 地理分布和传疟作用研究[J]. 中国媒介生物学及控制杂志, 1998, 9(6): 455-459.

ZHOU H N, LU Y R. Studies on geographical distribution, ecology and habits, role in malaria transmission of Anopheles dirus in Yunnan[J]. Chin J Vector Biol Control, 1998, 9(6): 455-459.

RAIKHEL A S, KOKOZA V A, ZHU J, et al . Molecular biology of mosquito vitellogenesis: from basic studies to genetic engineering of antipathogen immunity[J ] . Insect Biochem Mol Biol, 2002, 32(10): 1275-1286. DOI: 10.1016/s0965-1748(02)00090-5.

RAIKHEL A S, DHADIALLA T S. Accumulation of yolk proteins in insect oocytes[J]. Annu Rev Entomol, 1992, 37: 217-251. DOI: 10.1146/annurev.en.37.010192.001245.

NIRMALA X, MARINOTTI O, SANDOVAL J M, et al . Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi[J ] . Insect Biochem Mol Biol, 2006, 36(9): 694-700. DOI: 10.1016/j.ibmb.2006.05.011.

ISOE J, HAGEDORN H H. Mosquito vitellogenin genes: Comparative sequence analysis, gene duplication, and the role of rare synonymous Codon usage in regulating expression[J]. J Insect Sci, 2007, 7: 1-49. DOI: 10.1673/031.007.0101.

于莎莎, 王英, 李秋霞, 等. 大劣按蚊卵黄蛋白原cDNA的克隆及转录水平研究[J]. 中国热带医学, 2017, 17(4): 336-339.

YU S S, WANG Y, LI Q X, et al . Molecular cloning and transcription research of vitellogenin c DNA in Anopheles dirus[J ] . China Trop Med, 2017, 17(4): 336-339.

MARCHLER-BAUER A, BO Y, HAN L Y, et al . CDD/SPARCLE: functional classification of proteins via subfamily domain architectures[J ] . Nucleic Acids Res, 2017, 45(D1): D200-D203. DOI: 10.1093/nar/gkw1129.

任增亮, 堵国成, 陈坚, 等. 大肠杆菌高效表达重组蛋白策略[J]. 中国生物工程杂志, 2007, 27(9): 103-109.

REN Z L, DU G C, CHEN J, et al . Strategies for high-level expression of recombinant protein in Escherichia coli[J ] . China Biotechnol, 2007, 27(9): 103-109.

TELFER W H. Immunological studies of insect metamorphosis[J]. J Gen Physiol, 1954, 37(4): 539-558. DOI: 10.1085/jgp.37.4.539.

PAN M L, BELL W J, TELFER W H. Vitellogenic blood protein synthesis by insect fat body[J]. Science, 1969, 165(3891): 393-394. DOI: 10.1126/science.165.3891.393.

PIULACHS M D, GUIDUGLI K R, BARCHUK A R, et al . The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies[J ] . Insect Biochem Mol Biol, 2003, 33(4): 459-465. DOI: 10.1016/s0965-1748(03)00021-3.

VALLE D. Vitellogenesis in insects and other groups: a review[J]. Mem Inst Oswaldo Cruz, 1993, 88(1): 1-26. DOI: 10.1590/s0074-02761993000100005.

张士璀, 孙旭彤, 李红岩. 卵黄蛋白原研究及其进展[J]. 海洋科学, 2002(7): 32-35.

ZHANG S Y, SUN X T, LI H Y. Review on Vitellogenin[J]. Mar Sci, 2002 (7): 32-35.

李兆杰, 杨丽君, 王静, 等. 卵黄蛋白原的研究进展[J]. 生命科学, 2010,22(3): 284-290.

LI Z J, YANG L J, WANG J, et al . The progress in studies on vitellogenin[J ] . Chin Bull Life Sci, 2010, 22(3): 284-290.

罗永莉, 朱宏宏, 江艾耘, 等. 犬弓首蛔虫卵黄原蛋白DUF1943结构域的克隆及原核表达[J]. 西南大学学报(自然科学版), 2018(1): 15-20.

LUO Y L, ZHU H H, JIANG A Y, et al . Cloning and prokaryotic expression of the DUF1943 domain of Toxocara canis vitellogenin[J ] . J Southwest Agric Univ, 2018(1): 15-20.

TUFAIL M, TAKEDA M. Molecular characteristics of insect vitellogenins[J]. J Insect Physiol, 2008, 54(12): 1447-1458. DOI: 10.1016/j.jinsphys.2008.08.007.

CHEN J S, CHO W L, RAIKHEL A S. Analysis of mosquito vitellogenin cDNA. Similarity with vertebrate phosvitins and arthropod serum proteins[J]. J Mol Biol, 1994, 237 (5): 641-647. DOI: 10.1006/jmbi.1994.1261.

边长玲, 龚正达. 我国蚊类及其与蚊媒病关系的研究概况[J]. 中国病原生物学杂志, 2009, 4(7): 545-551.

BIAN C L, GONG Z D. Mosquitoes and mosquito-borne diseases in China[J]. J Pathog Biol, 2009, 4(7): 545-551.

WANG D, LI S, CHENG Z, et al . Transmission risk from imported Plasmodium vivax malaria in the China-Myanmar border region[J ] . Emerg Infect Dis, 2015, 21(10): 1861-1864. DOI: 10.3201/eid2110.150679.

霍岩, 陈晓英, 方荣祥, 等. 卵黄蛋白原的产生及其非营养功能的研究现状[J]. 生物技术通报, 2018, 34 (02): 66-73.

HUO Y, CHEN X Y, FANG R X, et al . Study on the production of vitellogenin and its non-nutritional functions[J ] . Biotech Bull, 2018, 34 (2): 66-73.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Cloning and bioinformatic analysis of cDNA fragment of TEP1 gene of Anopheles dirus

Cross-immunoreactivity between serine proteases from Anopheles dirus and Manduca

Related Author

胡晓飞

王英

潘春江

刘涛

谢佳

何谐

姜晓梅

黄复生

Related Institution

No data

⁰

Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain

DOI：10.16016/j.1000-5404.202012233

摘要

Abstract

关键词

Keywords

references