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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[1]	DUFOUR S, DUROCHER J, DUBUC J, et al. Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein-based ELISA and of transrectal ultrasonographic exam for diagnosis of pregnancy at 28-45 days following breeding in dairy cows [J]. Preventive Veterinary Medicine, 2017, 140: 122−133. DOI: 10.1016/j.prevetmed.2017.03.008
[2]	ZOLI A P, GUILBAULT L A, DELAHAUT P, et al. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy Diagnosis1 [J]. Biology of Reproduction, 1992, 46(1): 83−92. DOI: 10.1095/biolreprod46.1.83
[3]	PIECHOTTA M, BOLLWEIN J, FRIEDRICH M, et al. Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows [J]. Journal of Reproduction and Development, 2011, 57(1): 72−75. DOI: 10.1262/jrd.10-022T
[4]	KAREN A, DE SOUSA N M, BECKERS J F, et al. Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle [J]. Animal Reproduction Science, 2015, 159: 31−37. DOI: 10.1016/j.anireprosci.2015.05.005
[5]	GREEN J A, XIE S C, QUAN X, et al. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during Pregnancy1 [J]. Biology of Reproduction, 2000, 62(6): 1624−1631. DOI: 10.1095/biolreprod62.6.1624
[6]	WOODING F B P, ROBERTS R M, GREEN J A. Light and Electron microscope immunocytochemical studies of the distribution of pregnancy associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications [J]. Placenta, 2005, 26(10): 807−827. DOI: 10.1016/j.placenta.2004.10.014
[7]	HUGHES A L, GREEN J A, GARBAYO J M, et al. Adaptive diversification within a large family of recently duplicated, placentally expressed genes [J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(7): 3319−3323. DOI: 10.1073/pnas.97.7.3319
[8]	NAGAPPAN M, MICHAEL M, ROBERT S. Methods for early detection of pregnancy in cows: USA, PAT-US7604950[P]. 2009-10-20.
[9]	EL AMIRI B, REMY B, MELO DE SOUSA N, et al. Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation [J]. Reproduction Nutrition Development, 2004, 44(3): 169−181. DOI: 10.1051/rnd:2004025
[10]	BELLA A, SOUSA N M, DEHIMI M L, et al. Western analyses of pregnancy-associated glycoprotein family (PAG) in placental extracts of various mammals [J]. Theriogenology, 2007, 68(7): 1055−1066. DOI: 10.1016/j.theriogenology.2007.08.002
[11]	KLISCH K, DE SOUSA N M, BECKERS J F, et al. Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy [J]. Molecular Reproduction and Development, 2005, 71(4): 453−460. DOI: 10.1002/mrd.20296
[12]	GREEN J A, PARKS T E, AVALLE M P, et al. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers [J]. Theriogenology, 2005, 63(5): 1481−1503. DOI: 10.1016/j.theriogenology.2004.07.011
[13]	PATEL O V, TAKAHASHI T, IMAI K, et al. Generation and purification of recombinant bovine pregnancy associated glycoprotein [J]. The Veterinary Journal, 2004, 168(3): 328−335. DOI: 10.1016/j.tvjl.2003.09.012
[14]	薄小辉. 妊娠相关糖蛋白在奶牛早期妊娠诊断上的应用研究[D]. 北京: 中国农业科学院, 2017. FU X H. Application of pregnancy-related glycoproteins in the diagnosis of early pregnancy in cows[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017.
[15]	刘长彬, 卢春霞, 杨华, 等. 牛妊娠相关糖蛋白1(bPAG1)的真核表达及纯化[J]. 西南农业学报, 2019, 32(8): 1944-1949. LIU C B, LU C X, YANG H, et al. Eukaryotic expression and purification of recombinant bovine pregnancy Associated Glycoprotein-1[J]. Southwest China Journal of Agricultural Sciences, 2019, 32(8): 1944-1949. （in Chinese）
[16]	LIU C B, LU C X, SHI G Q. Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4 [J]. Analytical and Bioanalytical Chemistry, 2020, 412(18): 4235−4243. DOI: 10.1007/s00216-020-02666-w
[17]	刘长彬, 石国庆, 卢春霞. 牛妊娠相关糖蛋白9(bPAG9)的真核表达及纯化 [J]. 新疆农业科学, 2019, 56(8):1552−1559. LIU C B, SHI G Q, LU C X. Eukaryotic expression and purification of bovine pregnancy associated glycoprotein-9(bPAG9) [J]. Xinjiang Agriculture Science, 2019, 56(8): 1552−1559.(in Chinese)
[18]	MANSOURI M, MOUSAVY S J, EHSAEI Z, et al. The Codon-optimization of cfaE gene and evaluating its high expression capacity and conserved immunogenicity in Escherichia coli [J]. Biologicals: Journal of the International Association of Biological Standardization, 2013, 41(3): 169−175.
[19]	KIANMEHR A, GOLAVAR R, ROUINTAN M, et al. Cloning and expression of Codon-optimized recombinant darbepoetin Alfa in Leishmania tarentolae T7-TR [J]. Protein Expression and Purification, 2016, 118: 120−125. DOI: 10.1016/j.pep.2015.10.013
[20]	ŠNAJDER M, MIHELIČ M, TURK D, et al. Codon optimisation is key for pernisine expression in Escherichia coli [J]. PLoS One, 2015, 10(4): e0123288. DOI: 10.1371/journal.pone.0123288
[21]	GURUPRASAD K, BLUNDELL T L, XIE S, et al. Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases [J]. Protein Engineering, 1996(9): 849−856.

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