Construction of an Escherichia coli expression vector for the non-structural (NS)-1 protein of avian influenza virus H5N1 (Pembangunan vektor pengekspresan Escherichia coli untuk protein non-struktural (NS)-1 virus influenza unggas H5N1)
https://doi.org/10.17576/jskm-2020-1802-08
- Author:
ISTIQOMAH AGUSTA
1
;
JACINTA SANTHANAM
2
;
YAP WEI BOON
3
Author Information
1. Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jalan Salemba Raya No. 6, Jakarta Pusat 10430, DKI Jakarta, Indonesia
2. Program of Biomedical Science, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan Kuala Lumpur, Malaysia
3. Center of Toxicology and Health Risk Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan Kuala Lumpur, Malaysia
- Publication Type:Journal Article
- Keywords:
NS1;
avian influenza;
H5N1;
vaccine;
protein expression
- From:Malaysian Journal of Health Sciences
2020;18(No.2):73-81
- CountryMalaysia
- Language:English
-
Abstract:
In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.
- Full text:14.2020my0359.pdf
