Codon Optimization and Expression of Bovine Pregnancy-related Glycoprotein-16 Gene

LIU Changbin; LU Chunxia; LU Shouliang; NI Jianhong

doi:10.19303/j.issn.1008-0384.2021.01.001

Volume 36 Issue 1

Jan. 2021

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Fujian Journal of Agricultural Sciences > 2021 > 36(1): 1-8. > DOI: 10.19303/j.issn.1008-0384.2021.01.001

LIU C B, LU C X, LU S L, et al. Codon Optimization and Expression of Bovine Pregnancy-related Glycoprotein-16 Gene [J]. Fujian Journal of Agricultural Sciences，2021，36（1）：1−8. DOI: 10.19303/j.issn.1008-0384.2021.01.001

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Codon Optimization and Expression of Bovine Pregnancy-related Glycoprotein-16 Gene

1.
Key Laboratories of Sheep Breeding and Reproduce/Xinjiang Academy of Agriculture and Reclamation Science, Shihezi, Xinjiang 　832000, China
2.
School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing　408100, China

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Received Date: July 19, 2020
Revised Date: October 28, 2020
Available Online: February 07, 2021

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Abstract

Abstract

Objective The bPAG16 gene related to bovine pregnancy was codon-optimized and synthesized to construct recombinant vector expressed in HEK293 cells for the development of a diagnostic tool on pregnancy of dairy cows.
Methods Bioinformatics were applied to codon-optimize and synthesize the bPAG16 gene followed by connecting it to the proEM vector by T4 DNA ligase. The resulting proEM-bPAG16 recombinant vector was transfected into HEK293 cells. The expressed gene was purified by Ni²⁺ affinity chromatography and verified by SDS-PAGE and western blotting.
Result With the optimization, the codon adaptation index (CAI) of the bPAG16 gene rose from 0.77 to 0.96 and the GC content from 48% to 58%. After enzymatic digestion, fragments of approximately 1 179 bp and 4 369 bp were obtained from the proEM-bPAG16 expression vector as expected. The nucleotide sequence of the inserted bPAG16 in the recombinant plasmid was same as that of the optimized gene and absent of any mutated amino acids. SDS-PAGE and western blotting showed that the 48kDa recombinant bPAG16 fusion protein was correctly expressed in the HEK293 cells with a purity greater than 90%.
Conclusion The successfully executed codon optimization and synthesis of bPAG16 gene provided the base material for further study on early diagnosis of pregnancy in dairy cows.
- Pregnancy-related glycoprotein,
- codon optimization,
- recombinant protein,
- dairy cattle,
- early pregnancy diagnosis

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