鲫鱼肌间刺形成基因SOST的克隆表达研究
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摘要
硬化蛋白(Sclerostin,SOST)是一种由骨细胞特异性分泌用于负调节骨形成的因子,为探究硬化蛋白SOST在鲫鱼肌间刺形成中的调控作用.本研究对鲫鱼SOST基因进行扩增,克隆至原核表达载体pET-32a(+),构建重组质粒pET-32a(+)-SOST,转化感受态细胞BL21(DE3),利用IPTG诱导蛋白表达,分析诱导时间与诱导浓度对SOST蛋白表达的影响,并比较SOST基因内源信号肽片段切除前后的表达效果.结果显示,鲫鱼SOST基因序列为636 bp,蛋白质分子量约为38 kDa.随着时间的递增,蛋白表达量逐渐增大,诱导时间为4 h左右表达量达到最高;不同IPTG诱导浓度对蛋白的表达效果影响不大,而信号肽的存在会强烈抑制该蛋白的表达.研究结果为进一步分析SOST基因在鲫鱼肌间刺形成过程中的影响提供理论依据.
The sclerostin(SOST)is a factor that is specifically secreted by bone cells for negative regulation of bone formation. In order to explore the regulatory function of the gene SOST during the intermuscular thorn formation of Carassius auratus, we cloned the gene and constructed the prokaryotic expression vector pET-32 a(+)-SOST successfully. Then the recombinant plasmids were transferred into E.coli BL21(DE3)and induced by IPTG to count the protein expression. Meanwhile, using the SOST gene without the endogenous signal peptide, we check the protein expression again. It is respectably reflected that the length of the gene is about 636 bp and its protein weight is 38 kDa. Additionally, the induction of time did have an influence on the protein expression. After the SDS-PAGE, we found that the exist of endogenous signal peptide strong inhibits to protein prokaryotic expression. What's more, the concentration of IPTG has little effect on it while the protein expression became stable after 4 h induction in the time gradient. This experiment can provides more theoretical evidence for further analysis about SOST gene on the Carassius auratus intermuscular thorns formation mechanism.
The sclerostin(SOST)is a factor that is specifically secreted by bone cells for negative regulation of bone formation. In order to explore the regulatory function of the gene SOST during the intermuscular thorn formation of Carassius auratus, we cloned the gene and constructed the prokaryotic expression vector pET-32 a(+)-SOST successfully. Then the recombinant plasmids were transferred into E.coli BL21(DE3)and induced by IPTG to count the protein expression. Meanwhile, using the SOST gene without the endogenous signal peptide, we check the protein expression again. It is respectably reflected that the length of the gene is about 636 bp and its protein weight is 38 kDa. Additionally, the induction of time did have an influence on the protein expression. After the SDS-PAGE, we found that the exist of endogenous signal peptide strong inhibits to protein prokaryotic expression. What's more, the concentration of IPTG has little effect on it while the protein expression became stable after 4 h induction in the time gradient. This experiment can provides more theoretical evidence for further analysis about SOST gene on the Carassius auratus intermuscular thorns formation mechanism.
引文
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[2] 马良骁,董在杰,苏胜彦,等.鱼类肌间刺的研究进展[J].江苏农业科学,2012,40(4):234-235.
[3] 吕耀平,鲍宝龙,蒋燕,等.低等真骨鱼类肌间骨的比较分析[J].水产学报,2007,31(5):661-668.
[4] 董在杰,黄代中,李丽娟,等.几种常见鲤科养殖鱼类肌间刺的初步研究[J].上海水产大学学报,2006,15(4):425-429.
[5] 房连聪.淇河鲫肌间骨及其形成相关基因SOST的研究[D].郑州:河南师范大学,2015.
[6] 王良炎,田雪,庞小磊,等.硬化蛋白基因在淇河鲫成鱼不同肌间骨相邻肌组织的表达差异分析[J].中国生物化学与分子生物学报,2016,32(12):1354-1359.
[7] 秦龙娟,丁达霞,崔璐璐,等.SOST基因的表达调控[J].遗传,2013,5(8):939-947.
[8] 田雪,王良炎,陈琳,等.SOST基因在淇河鲫肌间骨骨化过程中的表达研究[J].水产学报,2016,40(5):673-680.
[9] 李雁飞,张金祥,谷艳芹,等.信号肽对OPMA在枯草杆菌中的分泌表达及其催化活性的影响[J].高等学校化学学报,2013,34(10):2334-2339.
[10] 赵慧,郑文岭,马文丽.信号肽对外源蛋白分泌效率的影响[J].生物学杂志,2003,20(5):1-3.
[11] 韦雪芳,王冬梅,刘思,等.信号肽及其在蛋白质表达中的应用[J].生物技术通报,2006(6):38-42.
[12] 李军,李伯良.蛋白A信号肽引导的E.coli外泌高表达异源蛋白[J].生物化学与生物物理学报,1995,27(6):616-623.
[13] 龚婷,杨孝朴,李银聚,等.鸡IL-2全基因和去信号肽基因的原核表达[J].甘肃农业大学学报,2009,44(1):11-14.
[14] 徐栋生,陈蕴,金坚.信号肽对肝细胞生长因子HGF在CHO中表达及分泌的影响[J].中国细胞生物学学报,2016,38(12):1473-1479.