[Expression of recombinant human beta-defensin-2 gene with C terminal of double marks of Myc and poly-histidine in transfected COS-7 cells].
The aim of this study is to explore the possibility and technical itinerary of establishing an mammal engineering cell line in which hBD-2 can be effectively expressed, secreted, detected, separated and purified. The full hBD2 cDNA was inserted into the multi clone site of a eukaryotic expressive plasmid pcDNA3.1/Myc-His(+) and located closely at the upstream of two tag gene (myc and 6 Poly-histidines) so as to construct another recombinant eukaryotic expressive vector of hBD-2 gene: rpcDNA3.1/Myc-His/hBD-2. By the use of RT-PCR with special primers, a band of 240 bp was amplified from COS-7 cells transfected by this recombinant plasmid, which matched full length of cDNA coding hBD2 plus myc epitope and 6 poly-histidines tags. Western blot analysis with specific anti-histidines antibody revealed that the lysate of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2 had a strong band with molecular weight of about 10 Kd that was approximate to the size of chiasmic peptide. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2.
Cai
Shaohui
S
Research Unit of Infection and Immunity, West China Medical Center of Sichuan University, Chengdu 610041.
Du
Jun
J
Cheng
Xinnian
X
Huang
Ning
N
Fumihiko
Nagase
N
Takaiki
Hasegawa
H
Wang
Boyao
B
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D015870:Gene Expression; D016259:Genes, myc; D006639:Histidine / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
C106641:DEFB4A protein, human; D023083:beta-Defensins; D006639:Histidine