cDNA Cloning, Prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide ResponseReportar como inadecuado




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Aim: To clone two splicing products of the mouse mLRG-cDNA and to express mLRG protein. Methods: The sequence obtained was compared human lrg to mouse genome with a comparative BLAST genome search and found completely identical. We spliced some fragments to a whole mouse lrg-cDNA sequence and designed a pair of primers at completely homologous fragments in 5’-UTR and 3’-UTR, we amplified mouse lrg-cDNA by RT-PCR. Then the sequence encoding the mLRG protein was amplified by RT-PCR from the total RNA of NIH3T3 cell stimulated by lps lipopolysaccharide, and we got two splicing products of mLRG mLRGW, mLRGS and two sequences encoding protein were cloned into the prokaryotic expression vector pTAT so as to construct the recombinant expression vector pTAT-MLRGW and pTAT-MLRGS. The proteins were expressed in E. coli BL21 DE3. Results: We got a cDNA fragment with the length of 1905 bp. Its location is at chromosome X qF4 site and we amplified two encoding regions covered 1554 bp and 1404 bp respectively mlrgW mlrgS. His-TAT-mLRGW and His-TAT-mLRGS fusion protein were expressed successfully. mlrgW is consist of 10 exons and 9 introns; mlrgS is consist of 11exons and 10 introns. Conclusion: Cloning of two splicing products of mouse novel gene MLRG and prokaryotic protein expressions are of help in the further study of this gene.

KEYWORDS

mLRG, Lipopolysaccharide Response Gene, cDNA Cloning, Prokaryotic Protein Expression

Cite this paper

Dai, Z. , Nie, Z. , He, L. , Guan, L. and Yang, Y. 2015 cDNA Cloning, Prokaryotic Expression of Two Splicing Products of mLRG, a Mouse Gene of Lipopolysaccharide Response. International Journal of Clinical Medicine, 6, 867-875. doi: 10.4236-ijcm.2015.611114.





Autor: Zhongming Dai1,2*, Zanguo Nie2*, Liang He3, Lina Guan4, Yunsheng Yang1

Fuente: http://www.scirp.org/



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