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Published on: September 10, 2017
[Cloning human heat shock protein 90beta-cDNA and constructing its eukaryon vector].
Tao Liu1, Jumei Zhao, Ling Tian
1Department of Otorhinolargngology, West China Hospital, Sichuan University, Chengdu 610041, China.
Summary
Researchers successfully cloned human heat shock protein 90 beta (HSP90beta) cDNA and created an expression vector for eukaryotic cells. This work facilitates further study of HSP90beta function in biological systems.
Area of Science:
- Molecular Biology
- Gene Cloning
- Recombinant DNA Technology
Background:
- Heat shock proteins (HSPs) play crucial roles in cellular protein homeostasis and stress response.
- HSP90beta is a significant member of the HSP90 family, involved in the conformational maturation of client proteins.
Purpose of the Study:
- To clone the complementary DNA (cDNA) of human heat shock protein 90 beta (hHSP90beta).
- To construct a functional eukaryotic expression vector for the cloned hHSP90beta gene.
Main Methods:
- Isolation of total RNA from human nasopharyngeal carcinoma (NPC) cells.
- Complementary DNA (cDNA) synthesis via reverse transcription-polymerase chain reaction (RT-PCR).
- Ligation of hHSP90beta cDNA into the pcDNA3.1(+) eukaryotic expression vector using restriction enzyme digestion and T4 DNA ligase, followed by transformation into E. coli XL1-blue and verification through double enzyme digestion and sequencing.
Main Results:
- Successful cloning of human HSP90beta cDNA.
- Construction and verification of the recombinant plasmid containing hHSP90beta inserted into the pcDNA3.1(+) vector.
- Confirmation of successful gene insertion via double enzyme digestion yielding fragments of expected sizes (5.4 kb and 2.1 kb).
Conclusions:
- The study successfully cloned human HSP90beta cDNA and constructed a eukaryotic expression vector.
- The developed vector is suitable for expressing the hHSP90beta gene in eukaryotic systems.
- This achievement provides a valuable tool for investigating the biological functions of human HSP90beta.
