GHRHR-SV1抗体的制备及其蛋白在黑色素瘤细胞中的表达

doi:10.13481/j.1671-587x.20160637

Abstract

Abstract:

Objective: To prepare the polyclonal antibody against growth hormone-releasing hormone receptor splice variant 1 (GHRHR-SV1),and to detect the expressions of GHRHR-SV1 protein in melanoma cells,and to lay a foundation for further study of the pathogenesis of tumor.Methods: The gene sequence of GHRHR-SV1 was designed and synthesized,and then cloned into pET-32 a (+)plasmid to construct the recombinant vector pET-GHRHR-SV1. After identification by PCR and sequencing,the recombinant plasmid was transformed into E.coli BL21 (DE3). Then 0,0.5,1.0,1.5, and 2.0 mmol·L^-1 isopropyl β-D-1-thiogalactopyranoside (IPTG) were added respectively to induce for 1,2,4, and 6 h at 37℃,30℃, and 25℃.The optimal dosage,time and temperature of IPTG of the target protein were observed.The protein was expressed under the optimal condition and purified by affinity chromatography.The polyclonal antibody was obtained by immunizing the rabbit with the purified protein.The titer was assayed by ELISA method.The expression of GHRHR-SV1 protein in B16-F10 cells was detected by Western blotting method.Results: The recombinant vector pET-GHRHR-SV1 was constructed successfully.The expression amount of GHRHR-SV1 protein was the highest when it was induced by 2.0 mmol·L^-1 IPTG for 2 h at 25℃. The relative molecular weight of induced protein was 24 000.After purification,the purity of protein was 80%.The GHRHR-SV1 antibody titer was 1:1 600 000.The GHRHR-SV1 protein was expressed in B16-F10 cells.Conclusion: The recombinant vector pET-GHRHR-SV1 is constructed successfully. The expression amount of GHRHR-SV1 protein in the condition of 2.0 mmol·L^-1 IPTG, 2 h-induction and at 25℃ is the highest.The high titer of ployclonal antibody is obtained.The GHRHR-SV1 protein can express in melanoma cells.

Key words: growth hormone-releasing hormone receptor splice variant 1, recombinant vector, melanoma, antibody, recombinant protein

CLC Number:

GUO Xiaolan, TAN Yin, CHEN Wensheng, LIN Yuyin, DAI Jianwei. Preparation of GHRHR-SV1 antibodyand expression ofits protein in melanoma cells[J].Journal of Jilin University Medicine Edition, 2016, 42(06): 1237-1242.

References

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Preparation of GHRHR-SV1 antibodyand expression ofits protein in melanoma cells

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