Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2
10.16250/j.32.1374.2017245
- VernacularTitle:pEGFP-N1-HBsAg-ROP2多基因重组表达质粒的构建及表达鉴定
- Author:
Rong MA
1
;
Ting XIAO
;
Jin LI
;
Hui SUN
;
Chao XU
;
Bing-Cheng HUANG
;
Kun YIN
;
Gui-Hua ZHAO
;
Yong CUI
;
Song ZHU
;
Gong-Zhen LIU
;
Ge YAN
;
Qing-Kuan WEI
Author Information
1. 济南大学山东省医学科学院医学与生命科学学院
- Keywords:
Toxoplasmosis;
Hepatitis B;
pEGFP-N1;
HBsAg;
ROP2;
Plasmid construction
- From:
Chinese Journal of Schistosomiasis Control
2018;30(2):184-188
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development. Methods According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites,the HBsAg gene was amplified by PCR.The HB-sAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene.The HBsAg-ROP2 fragment was amplified by PCR and digested with HindⅢand KpnⅠto clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2.The expression vector was transfected into HEK293T cells based on the identification of PCR amplifi-cation,restriction endonucleases and sequencing.Results The PCR product of HBsAg was about 700 bp,which was consis-tent with the theoretical value.Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3-HBsAg-ROP2 recombinant plasmid.The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment.The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank.The target plasmid was successfully transfect-ed into HEK293T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombi-nant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.
