整合型诱导表达猪生长激素（pGH）载体的构建及表达研究

doi:10.11843/j.issn.0366-6964.2013.07.003

Abstract

Abstract:

This study aimed to construct integrated porcine growth hormone (pGH) inducible expression vector and to validate the induction and expression efficiency at the cellular level. pTRE-GH fragment was amplified by PCR from pTRE-GH12 vector, then the ligation of the Sal I digestion fragment to the Sal I digestion vector pCAGGS-rtTA, the recombinant vector pCAGGS-rtTA-TRE-GH12（pTTGH）was constructed. The pTTGH vector DNA transfected porcine PK15 cells, and enriched with G418, pGH mRNA expression level was determined by QRT-PCR after adding induction substrate, with doxycycline (DOX) in culture medium. The expression change was observed by QRT-PCR and Western blot assay for pGH after different concentration of DOX inducing. The results of sequencing and restriction enzyme digestion showed that the pTTGH vector was constructed successfully. Compared with control cell groups, QRT-PCR results showed that pGH mRNA expression level was the highest at 36 h after DOX adding; within a certain range of the DOX concentration, the exogenous pGH mRNA expression level showed positive relationship with DOX concentration. Nevertheless, the pGH expression level showed no obvious relationship with DOX concentration and adding or not in normal cell groups. Results of gray intensity analysis showed that pGH protein level and a certain range of the DOX concentration in culture media had positive relationship. The results showed that the vector had been successfully constructed and which could realize pGH controllable expression. This study established a foundation for preparing pGH controllable expression transgenic animal and further studying on GH affection.

CLC Number:

S828
S813.3

JU Hui-ming, BAI Li-jing, JIANG Xing, LI Kui. Construction and Expression Study of Integrated pGH Inducible Expression Vector[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(7): 1008-1013.

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Construction and Expression Study of Integrated pGH Inducible Expression Vector

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