Construction and expression of eukaryotic expressing vector pCH510 of polypeptide CH50 and its chemotaxis and antitumor function by in vivo transfection.
To construct an eukaryotic expressing vector that expresses CH50, a recombinant Cell I-Hep II bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmid in vivo, the plasmid was constructed by DNA recombination. Gene transfection was performed in vitro and in vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfection in vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5'-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3'-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3. 1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells and in vivo in mouse. The expression of the plasmid in vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor.
Li
D
D
Department of Medical Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
Feng
Z
Z
Ye
S
S
Zhang
G
G
Zhang
H
H
Huang
B
B
Xiao
H
H
D016475:3T3 Cells; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002634:Chemotaxis, Leukocyte; D015941:Complement Hemolytic Activity Assay; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
C478562:CH50 recombinant polypeptide; D005353:Fibronectins; D011994:Recombinant Proteins