培养山羊痘病毒常用细胞在X-gal环境中的显色差异分析<sup>*</sup>

doi:10.13523/j.cb.2103070

中国生物工程杂志

2021, Vol. 41

Issue (9): 48-54 DOI: 10.13523/j.cb.2103070

技术与方法

培养山羊痘病毒常用细胞在X-gal环境中的显色差异分析^*

杨柳^1,²,牟豪^1,²,许国洋^1,²,白运川³,余远迪^1,^2,^***(

)

1 重庆市畜牧科学院重庆 402460
2 重庆市兽用生物制品工程技术研究中心重庆 402460
3 重庆酉阳土家族苗族自治县畜牧产业发展中心重庆 409812

Analysis of the Difference in Color Development of Cultured Goatpox Virus Common Cells in X-gal Environment

YANG Liu^1,²,MOU Hao^1,²,XU Guo-yang^1,²,BAI Yun-chuan³,YU Yuan-di^1,^2,^***(

)

1 Chongqing Academy of Animal Sciences, Chongqing 402460, China
2 Chongqing Research Center of Veterinary Biologicals Engineering and Technology, Chongqing 402460, China
3 Chongqing Youyang Tujia and Miao Autonomous County Animal Husbandry Development Center, Chongqing 409812, China

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摘要：

目的：为筛选出表达LacZ基因重组山羊痘病毒的适宜培养细胞系。方法：将常用于培养山羊痘病毒(GPV)的乳仓鼠肾细胞(BHK21)、羊肾细胞(SK)和羔羊睾丸细胞(LT)培养至单层,之后单层细胞用含X-gal的培养基继续培养,观察比较各细胞在X-gal环境中呈现的蓝色。提取单层细胞的RNA,进行LacZ基因的RT-PCR,回收PCR产物、连接载体、转化E.coli,挑阳性克隆测序分析。将单层细胞继续培养72 h,检测各细胞产生的β-半乳糖苷酶。结果：随着培养时间的延长,固体培养的BHK21、LT细胞孔中胶颜色变蓝且逐渐加深,显微镜观察可见细胞蓝斑,而SK细胞、空白、无X-gal孔中的胶未变色,镜检无细胞蓝斑;液体培养的单层细胞逐渐脱壁,培养液未见蓝色。RT-PCR产物电泳条带与预期分子量大小相符,测序结果显示目标DNA与LacZ基因序列相似性值达100%,表明3种细胞均有LacZ基因的转录本。β-半乳糖苷酶测定结果显示3种细胞均表达此酶,细胞产酶能力比较为BHK21>LT>SK,尤以SK细胞产β-半乳糖苷酶量极低。结论：显色差异法筛选出的羊肾细胞适宜培养表达LacZ基因的重组山羊痘病毒,结果可为山羊痘病毒新型疫苗研发提供一些参考。

关键词： LacZ基因; 山羊痘病毒; 显色差异法; 羊肾细胞

Abstract:

Objective:To screen out the suitable host cells for recombinant goat pox virus (GPV) expressing LacZ gene.Methods:The baby hamster syrian kidney cells (BHK21), sheep kidney cells (SK) and lamb testis cells (LT) commonly used for the culture of GPV were cultured to a single layer in the same cell plate. The monolayer cells were then cultured in the medium containing X-gal, and the blue color of each cell well in the environment of X-gal was observed. RNA was extracted from the monolayer cells and RT-PCR of LacZ gene was performed. PCR products were recovered, ligation vectors were transformed into E. coli (DH5α), and positive clones were selected for sequencing analysis. The β-galactosidase produced by each cell was tested when the monolayer cells were cultured for 72 h.Results:As the culture time prolonged, the gel color of BHK21 and LT wells in solid medium turned blue and deepened gradually. Blue spots were observed under microscope. But SK cells, blank and medium X-gal free did not change color, and no blue spots were observed under microscope. Besides the adherent cells in liquid culture containing X-gal gradually showed detachment, the culture medium was not blue. The electrophoresis band of the RT-PCR product was consistent with the expected molecular weight. The analysis of sequenced results showed that the DNA sequence similarity between the target DNA and the LacZ gene reached 100%, indicating that the 3 cells all have LacZ gene transcripts. The results of β-galactosidase assay showed 3 cells expressed this enzyme, and their production enzyme ability was BHK21>LT>SK, especially SK cells produced very low amount of β-galactosidase.Conclusion:The SK cell screened by the color difference method is suitable for culturing recombinant GPV expressing LacZ gene. The results can provide some reference value for the development of new GPV vaccine.

Key words: LacZ gene Goatpox virus Color difference method SK cell

收稿日期: 2021-03-29 出版日期: 2021-09-30

ZTFLH:

Q813

基金资助: * 重庆市技术创新与应用发展专项面上项目(cstc2019jscx-msxmX0325);农业部国家动物疫病长期性观测监测项目(18501)

通讯作者: 余远迪 E-mail: yuyuandi@126.com

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引用本文:

杨柳,牟豪,许国洋,白运川,余远迪. 培养山羊痘病毒常用细胞在X-gal环境中的显色差异分析^*[J]. 中国生物工程杂志, 2021, 41(9): 48-54.

YANG Liu,MOU Hao,XU Guo-yang,BAI Yun-chuan,YU Yuan-di. Analysis of the Difference in Color Development of Cultured Goatpox Virus Common Cells in X-gal Environment. China Biotechnology, 2021, 41(9): 48-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103070 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/48

图1 固定化培养的BHK21细胞、SK细胞、LT细胞在0 h、24 h、48 h和72 h的显色结果

图2 液体培养的BHK21细胞、SK细胞、LT细胞在24 h的显色观察与细胞生长状态

图3 固定化培养的BHK21细胞、SK细胞、LT细胞和空白在72 h的显微镜观察结果

图4 PCR产物的电泳图

图5 各细胞产β-半乳糖苷酶检测结果

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