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

doi:10.13523/j.cb.2103070

China Biotechnology

2021, Vol. 41

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

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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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

Received: 29 March 2021 Published: 30 September 2021

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Corresponding Authors: Yuan-di YU E-mail: yuyuandi@126.com

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Cite this article:

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.

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

Fig.1 The color development results of immobilized cell BHK21, SK, LT cultured at 0 h, 24 h, 48 h and 72 h

Fig.2 The color and cell adhensions of BHK21, SK, LT in liquid cultured at 24 h

Fig.3 Microscopic observation results of immobilized cell BHK21, SK, LT and blank group cultured at 72 h Arrows-cell blue spots

Fig.4 Electropherogram of PCR product M2000: DNA marker2000; 1: PCR amplified fragment of LacZ gene in recombinant positive plasmid; 2, 3, 4: RT-PCR amplified fragment of BHK21, SK, LT cell RNA as templates

Fig.5 The test results of β-galactosidase production on each cell cultured for 72 h Con: DMEN culture;*,** and *** represent P<0.05, P<0.01 and P<0.001, respectively


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