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Bioinformatics Analysis, Cloning and Expression of Spirometra erinaceieuropaei Fatty Acid-Binding Protein

Bioinformatics Analysis, Cloning and Expression of Spirometra erinaceieuropaei Fatty Acid-Binding Protein

Lin Huang1, Ling Mai2, Keyan Zhong1 and Xinjun Chen3*

1Experimental Animal Center for Teaching, Hainan Medical University, Haikou 571199, P.R. China.
2Department of Pathology, Danzhou People’s Hospital, Danzhou 571747, P.R. China.
3Laboratory of Pathogenic Biology and Immunology, Hainan Medical University, Haikou 571199, P.R. China.
 
Lin Huang and Ling Mai contributed equally to this work.
 
* Corresponding author: chenxj@hainmc.edu.cn

ABSTRACT

To clarify the characteristics of Spirometra erinaceieuropaei fatty acid-binding protein (SeFABP) and obtain its recombinant protein, the basic physio-biochemistry characteristics, signal peptides, antigen epitopes, transmembrane domains, secondary and tertiary structures, multi sequence alignment and molecular evolutionary tree of SeFABP were predicted and analyzed. On this basis, SeFABP was whole-genome synthesized and cloned into prokaryotic expression vector. The recombinant plasmid pET-30a(+)-SeFABP was constructed, then transformed into E. coli BL21 (DE3) and induced by IPTG. The purified SeFABP was obtained by Ni-IDA resins affinity chromatography, and were finally confirmed by SDS-PAGE and Western blot. The results showed that SeFABP, a stable intracellular protein without signal peptide and transmembrane region, was composed of 130 amino acids (AA). It contained 10 phosphorylation sites and 7 post-translational modification sites. Both methods predicted that SeFABP contained 5 linear epitopes, and were highly coincident, indicating that SeFABP had good immunogenicity. Its secondary structure contains 2 α-helix (13.07%), 10 β-sheet (56.92%) and 30% random coli. The comparison of multiple FABPs sequences showed that the species were identical and highly conserved at multiple AA sites. Specifically, SeFABP has the highest similarity with T. multiceps and M. vogae, close to 37%, but the lowest similarity with F. hepatica and F. gigatica, close to 22%. Plasmid double enzyme digestion and sequencing showed that the fully synthesized SeFABP fragment was accurately cloned and connected to the expression vector pET-30a(+)-SeFABP. After induction, the recombinant SeFABP was successfully expressed in the form of inclusion body. SDS-PAGE and Western blot showed a 15kD band, indicating that the recombinant was successfully purified. In conclusion, the characteristics of SeFABP and its high-purity recombinant protein were obtained, provides a basis for future research based on SeFABP in vaccine development and drug design.

 

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Pakistan Journal of Zoology

August

Vol. 54, Iss. 4, Pages 1501-2001

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