Construction and <em>in vitro</em> expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes

Xia Zhangquan; Zhang Congji; Wang Tao

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Construction and in vitro expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes

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- Construction and in vitro expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes
- Journal of Army Medical University Vol. 33, Issue 5, Pages: 463-468(2011)
- 作者机构：
  
  第三军医大学：西南医院口腔科，军事预防医学院防原医学教研室,全军复合伤研究所,创伤、烧伤与复合伤国家重点实验室
- 作者简介：
- 基金信息：
- DOI：
  CLC： R394.2;R394-33
- Published：2011，
  
  Published Online：15 March 2011，
- 稿件说明：
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XIA ZHANGQUAN, ZHANG CONGJI, WANG TAO. Construction and in vitro expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes. [J]. Journal of army medical university, 2011, 33(5): 463-468.
DOI：

XIA ZHANGQUAN, ZHANG CONGJI, WANG TAO. Construction and in vitro expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes. [J]. Journal of army medical university, 2011, 33(5): 463-468. DOI：

摘要

目的构建人血管内皮生长因子165(vascular endothelial growth factor

VEGF165) 和人β防御素3 (human beta defensins

HBD3 )双基因共表达真核质粒载体、观察其在293FT细胞中的表达

并检测其活性。方法利用RT-PCR方法分别从白血病细胞(HL60)和牙龈组织中扩增出VEGF165和HBD3基因片段

通过DNA重组技术将基因片段先后重组于pVIVO1-mcs真核表达载体上

构建成pVIVO1-VEGF165-HBD3重组质粒载体

分别用酶切电泳分析及DNA测序的方法对重组质粒载体进行鉴定。运用脂质体转染的方法将重组质粒转染293FT细胞后

RT-PCR和Western blot法检测目的基因的表达。用转染重组质粒的293FT无血清上清刺激内皮细胞EA.hy926增殖

CCK-8法检测其增殖效应。取转染重组质粒的293FT无血清上清的浓缩液，采用K-B纸片扩散法进行抗菌活性实验。结果 ①酶切电泳分析得到相应的目的片段

大小与理论计算值一致

DNA测序证实目的基因序列正确无突变

成功构建真核表达质粒载体pVIVO1-VEGF165-HBD3。②RT-PCR和Western blot法分别从mRNA和蛋白质2个水平证实：转染重组质粒的293FT细胞高表达VEGF165和HBD3。③转染重组质粒的293FT无血清上清明显促进内皮细胞EA.hy926的增殖，证实了表达分泌出的VEGF165的活性。转染重组质粒的293FT无血清上清的浓缩液对大肠埃希菌(

escherichia coli

EC)(ATCC 25922)、金黄色葡萄球菌(

staphylococcus aureus

SA)(ATCC 25923)、绿脓杆菌(

pseudomonas aeruginosa

PA)(ATCC 27853)和枯草芽孢杆菌(

bacillus subtilis

BS)(ATCC62037)产生明显的抑菌环，证实了表达分泌出的HBD3的活性。结论构建了pVIVO1-VEGF165-HBD3双基因共表达真核质粒载体

证实其转染293FT细胞后双基因的表达和活性。

Abstract

Objective To construct a bicistronic euk

aryotic expression plasmid carrying vascular endothelial growth factor (VEGF) 165 and human beta defensins (HBD) 3 genes

and to detect the expression and activity of VEGF165 and HBD3 in human embryonic kidney 293FT cells. Methods VEGF165 and HBD3 cDNA were amplified using total RNA from leukemic cell HL60 and gingival tissue through RT-PCR

respectively

and inserted into eukaryotic expression plasmid vector pVIVO1-mcs to construct bicistronic eukaryotic expression plasmid pVIVO1-VEGF165-HBD3. The plasmid was verified through restriction enzyme analysis and DNA sequencing and then transferred into 293FT cells through lipofection. VEGF165 and HBD3 expression in the transfected cells was detected through RT-PCR and Western blotting. Serum-free culture media supernatant of transfected 293FT cells was used to stimulate endothelial cell EA.hy926 proliferation

which was detected through the cell counting kit-8 (CCK-8) method. Antibacterial effects of the concentrated serum-free supernatant were detected via the Kleihauer-Betke (K-B) test. Results Restriction enzyme analysis and DNA sequencing results verified the successful construction of eukaryotic expression plasmid pVIVO1-VEGF165-HBD3. RT-PCR and Western blotting results exhibited high expression levels of VEGF165 and HBD3 in the transfected 293FT cells. The serum-free culture media supernatant can promote EA.hy926 cell proliferation significantly

reflecting the activity of secreted VEGF165. The concentrated serum-free supernatant could produce obvious inhibition rings against

Escherichia coli

(ATCC No. 25922)

Staphylococcus aureus

(ATCC No. 25923)

Pseudomonas aeruginosa

(ATCC No. 27853) and

Bacillus subtilis

(ATCC No. 62037)

showing the activity of secreted HBD3. Conclusion Bicistronic eukaryotic expression plasmid pVIVO1-VEGF165-HBD3 can express active VEGF165 and HBD3 in 293FT cells.

关键词

血管内皮生长因子165人β防御素3基因表达

Keywords

VEGF165HBD3gene expression

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Construction and in vitro expression of bicistronic eukaryotic expression plasmid carrying VEGF165 and HBD3 genes

DOI：

摘要

Abstract

关键词

Keywords

references