Construction of two retroviral vectors for interleukin-1 receptor antagonist and interleukin-10 and in vitro transfection of rabbit synoviocytes
- VernacularTitle:人白细胞介素-1受体拮抗剂或白细胞介素-10逆转录病毒载体的构建与体外实验
- Author:
Fei HAN
;
Ning ZHANG
;
Fulin TANG
- Publication Type:Journal Article
- Keywords:
Interleukin-1 receptor antagonist;
Interleukin-10;
Retroviridae;
Gene therapy
- From:
Chinese Journal of Rheumatology
2001;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objectives To construct two retroviral vectors, one containing human interleukin-1 recep-tor antagonist (hIL-1Ra) gene and the other containing human interleukin-10 (hIL-10) gene and to transfect rabbit synoviocytes in vitro and detect the expression level of target genes. Methods RNA from human peripheral blood mononuclear cells were extracted and target genes were amplified by RT-PCR. The target genes were cloned into retroviral vector pLXSN, which was then transducted into GP2-293 cells to produce recombinant retrovirus. Rabbit synoviocytes were transfected and the expression of target genes was detected by RT-PCR, immunohistochemistry and western-blot. Results The retroviral vector containing hIL-1Ra gene or hIL-10 gene was constructed successfully. The hIL-1Ra gene and hIL-10 gene were transduced respectively into rabbit synoviocytes in vitro. The mRNA level of both genes reached peak in 5 days. In positive cell clones, the protein level of hIL-1Ra reached peak within 30 days and maintained at least 60 days; the protein level of hIL-10 maintained at least 40 days. Conclusion The hIL-1Ra gene and hIL-10 gene can be transduced successfully into rabbit synoviocytes by recombinant retrovirus.
