VECTOR CONSTRUCTION FOR AFRICAN SWINE FEVER VIRUS CD2V ANTIGEN EXPRESSION ON BACULOVIRUS–INSECT CELL SYSTEM, USING THE Q5-SITE-DIRECTED MUTAGENESIS KIT

Nguyễn Thu Giang; Trần Ngọc Lân; Nguyễn Thị Thu Hiền; Lê Thị Thanh; Phạm Bích Ngọc

doi:10.34238/tnu-jst.9236

VECTOR CONSTRUCTION FOR AFRICAN SWINE FEVER VIRUS CD2V ANTIGEN EXPRESSION ON BACULOVIRUS–INSECT CELL SYSTEM, USING THE Q5-SITE-DIRECTED MUTAGENESIS KIT

About this article

Received: 15/11/23 Revised: 05/01/24 Published: 03/02/24

Authors

1. Nguyen Thu Giang, Institute of Biotechnology - Vietnam Academy of Science and Technology
2. Tran Ngoc Lan, University of Science and Technology of Hanoi
3. Nguyen Thi Thu Hien, Institute of Biotechnology - Vietnam Academy of Science and Technology
4. Le Thi Thanh , Institute of Biotechnology - Vietnam Academy of Science and Technology
5. Pham Bich Ngoc, Institute of Biotechnology - Vietnam Academy of Science and Technology

Abstract

African swine fever virus (ASFV) is the cause of African swine fever - one of the most dangerous infectious diseases in pigs with a 100% mortality rate. However, currently there is still no licensed vaccine for ASFV prevention. CD2v, a glycoprotein in the outer shell of ASFV, plays an important role in the process of virus infection and suppression of host immunity, and is a potential antigen for developing a subunit vaccine to prevent ASFV. In this study, the CD2v sequence of strain VNUAHY-ASFV/Vietnam2019 was optimized for expression on the baclovirus-insect cell system, artificially synthesized and cloned in the pUC57 vector. The entire CD2vFull-Foldon-His construct was cut and ligated into the pLIB vector to create the recombinant vector pLIB-CD2vFull-Foldon-His. Vector pLIB-CD2vFull-Foldon-His was used as a template to construct vector pLIB-CD2vED-Foldon-His carrying the trimeric CD2v extracellular domain, using the Q5 directed mutagenesis kit based on enzymatic PCR reaction with ADN Polymerase Q5 High-Fidelity. Next, vector pLIB-CD2vED-Foldon-His was used to design vector pLIB-CD2vED-His for expression of monomeric CD2vED using the Q5 kit also. Then, two vectors pLIB-CD2vED-Foldon-His and pLIB-CD2vED-His were transformed into E. coli DH10Bac to create recombinant bacmid for monomer/trimeric CD2vED expression on baculovirus-insect cell system for vaccine production against ASF.

Keywords

ASFV; CD2v ectodomain; Q5 directed mutagenesis kit; Baculovirus; Insect cell

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DOI: https://doi.org/10.34238/tnu-jst.9236

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