[Preparation and identification of monoclonal antibody against human testis development related gene 1].
OBJECTIVE
To construct a prokaryotic plasmid to express the testis development related gene 1 (TDRG1) recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, and to identify the biological properties of the mAb.


METHODS
The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, and then was expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtypes of mAb. Western blot and immunohistochemistry were used to detect specificity of mAb.


RESULTS
The prokaryotic plasmid expressing the recombinant protein was constructed, and the TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained. The titer of the mAb in ascites was 1 : 1.6 x 10(6), and the subtype of the mAb was IgG(1). Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.


CONCLUSIONS
The TDRG1 recombinant protein is highly purified and has strong antigenicity. The anti-TDRG1 mAb is produced successfully.
Wen
Jiaming
J
Department of Andrology, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Jiang
Xianzhen
X
Tang
Yuxin
Y
Yang
Jianfu
J
Chen
Houyang
H
Liu
Zhizhong
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013737:Testis / Q000166:cytology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018398:Homeodomain Proteins; D011994:Recombinant Proteins