[Prokaryotic expression of Bm86 gene of Boophilus microplus and optimization of the expression condition].
A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates, and subcloned into the prokaryotic plasmid pGEX-4T-1. The recombined plasmid was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time. SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST (Mr 94 000) after being induced with IPTG. High level expression of Bm86-GST was found at 1 mmol/L IPTG condition a fter incubation for 8 h at 37 degree C, and the expression level of the recombinant Bm86-GST reached up to 29% of total E coli proteins Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B. microplus positive serum.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Mi-Ling
ML
Ma
Gui-Quan
GQ
Guan
You-Quan
YQ
Li
Ai-Hong
AH
Liu
Qiao-Yun
QY
Ren
Qing-Li
QL
Niu
Hong
H
Yin
Jian-Xun
JX
Luo
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D015870:Gene Expression; D026863:Ixodidae / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014612:Vaccines / Q000235:genetics* / Q000276:immunology
C085065:BM86 protein, Boophilus; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D014612:Vaccines