转人干扰素α-2b细胞株建立及其外源基因表达分析

Establishment and exogenous genes expression analysis of cell strains secreted human interferon alpha 2b

【目的】从细胞水平探讨应用水牛乳腺生物反应器生产人干扰素α-2b(IFNα-2b)的可行性,为水牛乳腺生物反应器的制备提供参考依据。【方法】建立并优化核转染人乳腺癌细胞Bcap-37的方法,再利用该方法将水牛乳腺特异性表达人IFNα-2b载体导入Bcap-37细胞,建立转基因细胞株,然后分别从RNA水平和蛋白质水平对其能否正常表达人IFNα-2b进行分析。【结果】电压是影响Bcap-37细胞核转染效率的关键因素,对其进行优化(电压275 V、电击时间10 ms)后的核转染效率可达81.1%。使用优化后的核转染方法可将水牛乳腺特异性表达载体p BCN-IFN-CMVEGFP-neo和p BCN-IFN-CMV-EGFP-IRES-neo成功转入Bcap-37细胞而获得转人IFNα-2b细胞株,经IFNα-2b基因的m RNA表达丰度及IFNα-2b蛋白的Western blotting检测,证实在两株转基因细胞系中均能顺利进行IFNα-2b基因的转录与翻译。【结论】建立的核转染法能高效转染Bcap-37细胞,可应用该方法在细胞水平上对携带人IFNα-2b基因水牛乳腺特异性表达载体的表达能力进行分析,并证实乳腺特异性表达载体(p BCN-IFN-CMV-EGFP-neo和p BCN-IFN-CMVEGFP-IRES-neo)均能用于水牛乳腺生物反应器的制备。

[ObjectivelThe present study was conducted to investigate application of uffalo mammary gland bioreactor in production of human interferon alpha 2b （IFNα-2b） and the feasibility of that method at the cellular level, in order to provide reference for preparation of uffalo mammary gland bioreactor. [Method]The nucleofection method for transfecting Bcap-37 cells was established and optimized firstly. And then the buffalo mammary gland specific expressing vector was delivered into Bcap-37 cells, the transgenic cell were screened subsequently to establish transgenic cell strains. Finally, the IFNα-2b expression in transgenic cell strains was analyzed from the perspectives of RNA and protein expression level, respectively. [ Result ]The voltage was a key influencing factor of nuclofection efficiency, the nucleofection efficiency reached up to 81.1% under optimal condition （voltage of 275 V combined with electric shock time of 10 ms）. Using the optimized method of nucleofection, the buffalo mammary gland vectors pBCN-IFN-CMV-EGFP-neo and pBCN-IFN- CMV-EGFP-IRES-neo were transfected into Bcap-37 cells to obtain IFNα-2b cell strains. The IFNα-2b mRNA and pro- tein expression levels were detected by Western blotting, respectively, the detection results confirmed that IFNα-2b gene could be transcripted and translated correctly in two transgenic cell strains. [Conclusion]The established nucleofection method can be applied to transfect efficiently Bcap-37 cells to analyze expression level of gene buffalo mammary gland specific expressing vector carrying human IFNa-2b gene at the cellular level, which confirm that two buffalo mammary gland specific expressing vectors （pBCN-IFN-CMV-EGFP-neo and pBCN-IFN-CMV-EGFP-IRES-neo） can be used to nrenare uffalo mammarv aland bioreactor.