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[Prokaryotic expression of Bm86 gene of Boophilus microplus and optimization of the expression condition]
Mi-Ling Ma1, Gui-Quan Guan, You-Quan Li
1State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Abstract:
A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates, and subcloned into the prokaryotic plasmid pGEX-4T-1. The recombined plasmid was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time. SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST (Mr 94 000) after being induced with IPTG. High level expression of Bm86-GST was found at 1 mmol/L IPTG condition a fter incubation for 8 h at 37 degree C, and the expression level of the recombinant Bm86-GST reached up to 29% of total E coli proteins Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B. microplus positive serum.
Insights
Researchers successfully cloned and expressed the Boophilus microplus Bm86 gene in E. coli. The resulting Bm86-GST fusion protein was highly expressed and recognized by specific antibodies, aiding in tick vaccine development.
Area of Science:
- Molecular Biology
- Recombinant Protein Expression
- Immunology
Context:
- Boophilus microplus is a significant cattle tick pest.
- The Bm86 gene encodes a surface protein crucial for tick resistance.
- Developing effective vaccines against ticks is essential for livestock health.
Purpose:
- To clone and express the Bm86 gene from Boophilus microplus.
- To produce a recombinant Bm86-GST fusion protein in E. coli.
- To confirm the identity and expression level of the recombinant protein.
Summary:
- Specific primers were used to clone the Bm86 gene from Boophilus microplus.
- The cloned gene was subcloned into a prokaryotic expression vector (pGEX-4T-1) and transformed into E. coli BL21(DE3).
- High-level expression of the Bm86-GST fusion protein (Mr 94,000) was achieved using 1 mmol/L IPTG at 37°C for 8 hours, reaching 29% of total E. coli proteins. Western blotting confirmed the protein's identity using anti-B. microplus serum.
Impact:
- This study provides a foundation for developing subunit vaccines against Boophilus microplus.
- The successful expression of Bm86-GST facilitates further research into tick control strategies.
- The recombinant protein can be used for diagnostic and immunological studies related to cattle tick infestations.
