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Published on: June 15, 2018
Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia
Katayoun Dastan1, Mehdi Assmar1,2, Nour Amirmozafari1,3
1Department of Microbiology, Faculty of Basic Sciences, Lahijan Branch, Islamic Azad University, Lahijan, Iran.
Background:
Strongyloidiasis is a public health concern in northern regions of Iran, caused by Strongyloides stercoralis. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient's sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ELISA kit for the detection of antibodies against the nematode, IgG4 immunoreactive protein (NIE) was expressed in Escherichia coli expression system, purified and verified.
Methods:
The NIE gene sequence was retrieved from the GenBank. This sequence was codon-optimized for the expression in E. coli BL21 (DE3). The sequence was inserted into the expression vector pET-30b (+). The recombinant vector was then transferred into competent E. coli BL21 (DE3). Transformed colonies were selected and verified by colony PCR. NIE gene expression was induced with IPTG induction. The protein production was evaluated by SDS-PAGE and verified using Western blotting.
Results:
The codon-optimized NIE gene had required parameters for expression in E. coli. NIE protein was proved and verified by SDS-PAGE and Western blotting.
Conclusion:
NIE recombinant protein was successfully expressed in E. coli expression system in appropriate amounts. The recombinant protein can be used for developing ELISA kit in diagnosis of S. stercoralis.
