不同质粒载体对<i>GFP</i>单基因瞬时转染ExpiCHO-S细胞表达量的影响

王妍; 张超林; 王娟; 邓跃; 苏晓蕊; 谭菲菲; 田克恭

doi:10.19303/j.issn.1008-0384.2020.08.006

不同质粒载体对GFP单基因瞬时转染ExpiCHO-S细胞表达量的影响

doi: 10.19303/j.issn.1008-0384.2020.08.006

王妍^1,,
张超林²,
王娟²,
邓跃¹,
苏晓蕊²,
谭菲菲^2, ,,
田克恭^{1, 2, ,}

1.
河南农业大学牧医工程学院，河南　郑州　450046
2.
国家兽用药品工程技术研究中心，河南　洛阳　471003

基金项目: 河南省重大科技项目（181200211700）

详细信息

作者简介:
王妍（1994−），女，硕士研究生，研究方向：动物传染病（E-mail：869859241@qq.com）

通讯作者:
谭菲菲（1983−），女，博士，研究方向：CHO系统重组蛋白的表达、纯化及工艺研究（E-mail：tf0801@126.com）

田克恭（1964−），男，研究员，研究方向：动物疫病诊断检测与防治技术研究（E-mail：vetvac@126.com）

中图分类号: S 855.3
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出版历程
- 收稿日期: 2020-03-19
- 修回日期: 2020-07-07
- 刊出日期: 2020-08-19

Effects of Vectors on Transient Expression of GFP in CHO Cells

WANG Yan^1
,,
ZHANG Chaolin²,
WANG Juan²,
DENG Yue¹,
SU Xiaorui²,
TAN Feifei^{2
, ,},
TIAN Kegong^{1, 2
, ,}

1.
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan　450046, China
2.
National Research Center for Veterinary Medicine, Luoyang, Henan　471003, China

摘要

摘要: 目的 ExpiCHO细胞瞬时表达能够快速有效地生产重组蛋白，为筛选有效的重组蛋白节省了大量的时间，细胞系的选择、表达载体的选择、转染试剂的选择等是制约ExpiCHO细胞瞬时表达蛋白的重要因素，本研究旨在筛选出在CHO细胞中瞬时表达量较高的载体。方法以GFP基因作为目的基因，用不同的真核表达载体构建GFP重组质粒，瞬时转染ExpiCHO-S细胞。转染后4 d观察不同载体表达GFP的荧光数量和强度；转染后8 d对细胞进行裂解，收获裂解上清，用SDS-PAGE对不同载体的表达量进行鉴定。同时用His Trap FF亲和层析柱对裂解上清中的GFP蛋白进行纯化，通过Western Blot比较不同载体GFP蛋白的表达量。结果 pCDNA3.1-GFP、pCDNA3.4-GFP载体表达的荧光数量最多，pCIneo-GFP载体表达的荧光强度最强，pCMVHA载体表达的荧光数量最少，荧光强度最弱。SDS-PAGE和Western Blot结果均表明pCDNA3.1-GFP、pCDNA3.4-GFP、pCIneo-GFP重组质粒的蛋白表达量高于pCHO-GFP、pCMVHA-GFP重组质粒的蛋白表达量。结论筛选出表达量较高的真核表达载体pCDNA3.1、pCDNA3.4、pCIneo，为后续重组蛋白的瞬时表达载体选择提供依据。
- GFP /
- CHO细胞 /
- 真核表达载体
Abstract: Objective Appropriate cell line, expression vector, and transfection reagent were selected to enhance the transient expression of GFP recombinant protein for quick and efficient production of the proteins in CHO cells. Methods Targeting GFP, various eukaryotic vectors were employed to carry the gene protein for transfection into ExpiCHO-S cells. Four days after transfection, number and intensity of expression fluorescence on the recombinant plasmids by different vectors were recorded. Four more days later, the cells were lysed, and the lysate supernatant harvested to verify the gene expressions by SDS-PAGE. Meanwhile, GFP recombinant plasmids in the supernatant were purified on a His Trap FF affinity chromatography column to quantify the protein expressions by western blot. Results The recombinant plasmids, pCDNA3.1-GFP and pCDNA3.4-GFP had the expression fluorescence the greatest in number, pCIneo-GFP the highest on intensity, while pCMVHA the fewest in number and the lowest on intensity. SDS-PAGE and western blot showed the expressions of the first 3 recombinant plasmids were higher than those of pCHO-GFP or pCMVHA-GFP. Conclusion The GFP recombinant plasmids constructed with the eukaryotic vectors, pCDNA3.1, pCDNA3.4, and pCIneo, exhibited the greatest expressions and were considered the choice vectors for future studies requiring transient expression.
- GFP /
- CHO cells /
- eukaryotic expression vector

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图 1 不同载体构建GFP重组质粒双酶切结果

注：M1：DNA Marker；1：pCHO-GFP重组质粒酶切；2：pCDNA3.1-GFP重组质粒酶切；3：pCDNA3.4-GFP重组质粒酶切；4：pCIneo-GFP重组质粒酶切；5：pCMVHA-GFP重组质粒酶切；M2：DNA Marker。

Figure 1. Restriction map of GFP recombinant plasmids carried by different vectors

Note: M1: DNA marker; 1: digestion site for pCHO-GFP recombinant plasmid; 2: digestion site for pCDNA3.1-GFP recombinant plasmid; 3: digestion site for pCDNA3.4-GFP recombinant plasmid; 4: digestion site for pCIneo-GFP recombinant plasmid; 5: digestion site for pCMVHA-GFP recombinant plasmid; M2: DNA marker.

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图 2 不同载体表达GFP蛋白的荧光反应

Figure 2. Fluorescence of GFP expressed by different vectors

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图 3 SDS-PAGE分析转染后4 d GFP蛋白的表达

注：M：Protein Marker；1：ExpiCHO细胞裂解上清；2：pCDNA3.1-GFP细胞裂解上清；3：pCDNA3.4-GFP细胞裂解上清；4：pCIneo-GFP细胞裂解上清；5：pCHO-GFP细胞裂解上清；6：pCMVHA-GFP细胞裂解上清。

Figure 3. SDS-PAGE on GFP expression 4 d after transfection

Note: M: Protein marker; 1: ExpiCHO cell lysate supernatant; 2: cell lysate supernatant (pCDNA3.1-GFP); 3: cell lysate supernatant (pCDNA3.4-GFP); 4: cell lysate supernatant (pCIneo-GFP); 5: cell lysate supernatant (pCHO-GFP); 6: cell lysate supernatant (pCMVHA-GFP).

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图 4 Western Blot分析纯化后的GFP蛋白

注：1：ExpiCHO细胞裂解上清；2：GFP蛋白（pCMVHA）纯化后；3：GFP蛋白（pCHO）纯化后；4：GFP蛋白（pCIneo）纯化后；5：GFP蛋白（pCDNA3.4）纯化后；6：GFP蛋白（pCDNA3.1）纯化后；M：Protein Marker。

Figure 4. Western blot on purified GFP protein

Note: 1: ExpiCHO cell lysate supernatant; 2: purified GFP protein (pCMVHA); 3: purified GFP protein (pCHO); 4: purified GFP protein (pCIneo); 5: purified GFP protein (pCDNA3.4); 6: purified GFP protein (pCDNA3.1); M: Protein marker.

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表 1 不同载体的酶切位点及引物序列

Table 1. Digestion sites and primer sequences on different vectors

载体名 Vectors	上游引物序列 Sequence of upstream primers	下游引物序列 Sequence of downstream primers	上游酶切位点 Upstream restriction site	下游酶切位点 Downstream cleavage site
pCDNA3.1	CTAGCTAGCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATC	CGGCTCGAGTTAATGATGATGATGATGATGCTTGTACAGCTCATC	Nhe I	Xho I
pCDNA3.4	CTAGTCTAGAATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCAT	GGGAAGCTTTTAATGATGATGATGATGATGCTTGTACAGCTCATC	Xba I	Hind III
pCMVHA	CCGGAATTCGCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCA	CGGCTCGAGTTAATGATGATGATGATGATGCTTGTACAGCTCATC	Eco RI	Xho I
pCIneo	CTAGCTAGCATGGTGAGCAAGGGCGCCGAGCTGTTCACCGGCATC	CGGCTCGAGTTAATGATGATGATGATGATGCTTGTACAGCTCATC	Nhe I	Xho I
pCHO1.0	CCGCCTAGGGCCACCATGGTGAGCAAGGGCGCCGAGCTGTTCACC	CGGGTATACTTAATGATGATGATGATGATGCTTGTACAGCTCATC	Avr II	Bstz 17I
注：斜体碱基为酶切位点。 Note: The italic bases are restriction sites.

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表 2 不同载体片段大小

Table 2. Fragment sizes of different vectors

载体名称 Vectors	载体大小 Vectors size/bp
pCDNA3.1	5 333
pCDNA3.4	6 010
pCMVHA	3 782
pCIneo	5 747
pCHO	12 988

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表 3 不同载体表达GFP蛋白的荧光细胞数

Table 3. Number of fluorescent cells expressing GFP by different vectors

GFP蛋白表达载体 GFP protein expression vector	荧光细胞数量 The number of fluorescent cells
pCDNA3.1	168
pCDNA3.4	110
pCIneo	47
pCHO	151
pCMVHA	58

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表 4 不同载体表达GFP蛋白的灰度比值

Table 4. Gray ratio of vectors expressing GFP

GFP蛋白表达载体 GFP protein expression vector	灰度比值 Gray ratio
pCMVHA	1.00
pCHO	1.36
pCIneo	2.13
pCDNA3.4	2.68
pCDNA3.1	3.19

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