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Published on: August 14, 2021
[Recombinant plasmid constructed and cytotoxicity studied for outer membrane protein LipL32 gene of Leptospira strain
1Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To construct the recombinant plasmid containing the outer membrane protein LipL32 gene of Leptospira strain 017 and to study on the cytotoxicity of the expression protein.
Methods:
By the polymerase chain reaction (PCR), the LipL32 gene was amplified from Leptospira strain 017 genome and cloned into pET32a(+) with enzyme digestion, then used to transform E. coli JM109. After induced with IPTG, the target protein was expressed and used to immunize New zealand white rabbit. Western Blotting identified the immunogenicity of the expressed protein. Then the purified and renatured protein was acted on ECV304 cell so as to get its cytotoxicity detected by examining the LDH and NO (nitrogen monoxide) release from cell.
Results:
The full length of the LipL32 gene about 816 bp was obtained by PCR. The recombinant plasmid was identified by enzyme digestion, PCR and DNA sequencing. After induced with IPTG, the expressed protein existed mainly in the form of inclusion bodies about 52 x 10(3) (relative molecular mass) which was consistent with the expected size of the fused protein. After rabbit immunity, the titre of the produced multiclonal antibody reached 1 : 32 000 measured by ELISA. Western Blotting analysis found a positive band specifically in the target protein position. The release of the LDH and NO of the ECV304 cell treated with LipL32 had significant increase compared with the control group.
Conclusion:
The recombinant plasmid containing LipL32 gene is successfully constructed and can express the target protein in E. coli JM109. The expressed target protein has cytotoxicity.
Insights
Researchers constructed a recombinant plasmid with the Leptospira outer membrane protein LipL32 gene. The expressed LipL32 protein demonstrated significant cytotoxicity to ECV304 cells, evidenced by increased LDH and NO release.
Area of Science:
- Molecular Biology
- Microbiology
- Immunology
Background:
- Leptospira outer membrane protein LipL32 is a key virulence factor.
- Understanding LipL32's role in pathogenesis requires functional studies.
- Recombinant expression facilitates protein purification and functional analysis.
Purpose of the Study:
- To construct a recombinant plasmid encoding Leptospira strain 017 LipL32.
- To express and characterize the recombinant LipL32 protein.
- To evaluate the cytotoxicity of the expressed LipL32 protein.
Main Methods:
- Polymerase chain reaction (PCR) amplification and cloning of the LipL32 gene into pET32a(+).
- Bacterial transformation, protein expression induction (IPTG), and purification.
- Immunization of rabbits, antibody titration (ELISA), and Western Blotting.
- In vitro cytotoxicity assays on ECV304 cells measuring LDH and NO release.
Main Results:
- Successful construction and verification of the recombinant plasmid via enzyme digestion, PCR, and sequencing.
- Expression of the LipL32 fusion protein (approx. 52 kDa) primarily as inclusion bodies.
- High-titer polyclonal antibody production (1:32,000) confirmed by ELISA and Western Blotting.
- Significant increase in LDH and NO release from ECV304 cells upon treatment with purified LipL32 protein.
Conclusions:
- The recombinant plasmid containing the LipL32 gene was successfully constructed and expressed in E. coli.
- The expressed LipL32 protein exhibits cytotoxic effects on ECV304 cells.
