scholarly journals Cloning expression and immunogenicity analysis of inhibin gene in Ye Mule Aries sheep

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7761
Author(s):  
Zengwen Huang ◽  
Juan Zhang ◽  
WuReliHazi Hazihan ◽  
Zhengyun Cai ◽  
Guosheng Xin ◽  
...  
Keyword(s):  
Expression Vector ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Eukaryotic Expression ◽  
Eukaryotic Expression Vector ◽  
Restriction Sites ◽  
Polymerase Chain ◽  
Novel Strategy ◽  
Ovary Function ◽  
Potential Regulatory Role

Background Ye Mule Aries sheep is one of the most important sheep breeds in Xinjiang, China. This breed is well adapted to harsh environmental conditions and displays strong disease resistance, fast growth, and high cold tolerance. To analyze the clonal expression and immunogenicity of the Ye Mule Aries sheep inhibin gene, total RNA was extracted from sheep ovarian tissue and used as a template to generate a eukaryotic expression vector and study inhibin immunogenicity. Methods Primers were designed to amplify the inhibin A gene via polymerase chain reaction and the amplified product was cloned between the ScalI and EcoRI restriction sites of the expression vector pEGFP-N1 to construct a recombinant plasmid, pEGFP-INHα. Following the validation of successful cloning, the pEGFP-INHα plasmid was transfected into BHK cells to verify expression in eukaryotes and subsequently utilized as an antigen in rabbits. Rabbits were tested for anti-inhibin antibodies and serum follicle-stimulating hormone (FSH) concentrations. Results The analysis of the INHα gene sequence revealed that INHα is 1109 bp long and is translated to an approximately 40 KDa protein. Bioinformatics approach indicated that the INHα gene is highly conserved between organisms. Immunization with the eukaryotic expression vector, pEGFP-INHα, which expresses the INHα gene elicited immune response and generatigeneration on of anti-INHα antibody. The antibody had a significant regulatory effect on the serum concentration of FSH in rabbits and led to higher levels of FSH, indicating increased ovary function. Conclusions The present work resulted in a successful construction of eukaryotic expression plasmid pEGFP-INHα and verified the immunogenicity of this highly conserved protein. Further, the expression of pEGFP-INHα was shown to have a significant impact on the secretion of FSH, indicating a potential regulatory role in ovarian function. In conclusion, our current findings can serve as a working model for studying the effect of INHα on the breeding performance of Ye Mule Aries sheep, providing a novel strategy to improve their reproduction rates.

Construction of Eukaryotic Expression Vector and Transient Expression on Mycobacterium bovis mpb83,mpb70 and Cattle il-15 Gene

2013 ◽  
Vol 749 ◽  
pp. 177-181
Author(s):  
Yan Ru Zheng ◽  
Chun Fang Wang ◽  
Yun Hang Gao ◽  
Jia Ning Guan ◽  
Jia Ming Lin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Bovis ◽  
Recombinant Plasmid ◽  
Expression Vector ◽  
Fusion Gene ◽  
Eukaryotic Expression ◽  
Chain Reaction ◽  
Eukaryotic Expression Vector ◽  
Polymerase Chain ◽  
I Gene

Based on the splicing by overlapping extension (SOE) polymerase chain reaction (PCR),fusion gene mpb83-70 andil-15were ligated and cloned into pMD18-T,then recombinant plasmid pMD-83-70-15 was constructed. pMD-83-70-15 and pVAX1-BMS were digested by double enzymesHindIII andEcoRI, the purified mpb83-70-il-15 was subcloned into pVAX1-BMS,then recombinant plasmid pVAX1-BMS-83-70-15 was constructed and transient expressed in Mark145 cell. These results laid solid foundations for further studies on mpb83-70-il-15.

scholarly journals Experimental Study of the Effects of Marrow Mesenchymal Stem Cells Transfected with Hypoxia-Inducible Factor-1αGene

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Jinfu Yang ◽  
Tao Tang ◽  
Feng Li ◽  
Wenwu Zhou ◽  
Jian Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Expression Vector ◽  
Hypoxia Inducible Factor ◽  
Eukaryotic Expression ◽  
Eukaryotic Expression Vector ◽  
Hypoxia Inducible Factor 1 ◽  
Marrow Mesenchymal Stem Cells ◽  
Polymerase Chain

Objective.To construct the eukaryotic expression vector hypoxia-inducible factor 1α-pcDNA3.1and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-1αgene in MSCs.Methods.mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-1αwas amplified by polymerase chain reaction (PCR), and constructed intopcDNA3.1. Transfected HIF-1α-pcDNA3.1into MSCs by liposome mediated method. The expression of HIF-1αin the cells was detected by Western Blot Analysis and ELISA.Results.Eukaryotic expression vector HIF-1α-pcDNA3.1was constructed successfully. Analyzed by flow cytometer, The MSCs' surfaces mark were CD44+, SH3(CD73)+, CD34−, CD45−and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF-1αgene transfected MSCs, the expression of HIF-1αmRNA and HIF-1αprotein were both increased obviously.Conclusion.HIF-1αwas cloned successfully. HIF-1α-pcDNA3.1can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-1αin transfected MSCs.

Construction of Eukaryotic Expression Vector and Expression on Mycobacterium bovis ag85a and mpb70 Genes

2013 ◽  
Vol 421 ◽  
pp. 308-312
Author(s):  
Shuang Hou ◽  
Chun Fang Wang ◽  
Yan Ru Zheng ◽  
Jia Ning Guan ◽  
Jia Ming Lin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Bovis ◽  
Recombinant Plasmid ◽  
Expression Vector ◽  
Fusion Gene ◽  
Eukaryotic Expression ◽  
Chain Reaction ◽  
Eukaryotic Expression Vector ◽  
Polymerase Chain

Based on the polymerase chain reaction (PCR),ag85aandmpb70Fusion gene ofMycobacterium boviswere ligated and cloned into pMD18-T, then recombinant plasmid pMD-85a-70 was constructed. pMD-85a-70 and pVAX1-BMS were digested by double enzymesHindIII andEcoRI, the purified ag85a-mpb70 was subcloned into pVAX1-BMS, then recombinant plasmid pVAX1-BMS-85a-70 was constructed and transient expressed in Marc145 cell. These results laid solid foundations for further studies on ag85a-mpb70.

Construction and identification of recombinant eukaryotic expression vector pCI-VP7 containing GCRV VP7 gene

2010 ◽  
Vol 34 (5) ◽  
pp. 807-813
Author(s):  
Gui-jie HAO ◽  
Xiao-yi PAN ◽  
Jia-yun YAO ◽  
Yang XU ◽  
Wen-lin YIN ◽  
...  
Keyword(s):  
Expression Vector ◽  
Eukaryotic Expression ◽  
Eukaryotic Expression Vector ◽  
Vp7 Gene

scholarly journals Construction of a recombinant eukaryotic expression vector containing DNM3 gene and its expression in colon cancer cells

2018 ◽  
Vol Volume 11 ◽  
pp. 6665-6671
Author(s):  
Liang Jiang ◽  
Qi-Lian Liang ◽  
Wei-Ming Liang ◽  
Hui-Jie Zhang ◽  
Jie Huang ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Expression Vector ◽  
Eukaryotic Expression ◽  
Colon Cancer Cells ◽  
Eukaryotic Expression Vector ◽  
Em Gene
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