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[Expression of Proteus mirabilis polyphosphate kinase and preparation of its polyclonal antibodies]
Liang Peng1, Jing-Yi Ou, Jia-Yun Pan
1Department of Clinical Laboratory, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China.E-mail: pl_206@126.com.
Objective:
To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK.
Methods:
The antigenicity and hydrophobicity of PPK were analyzed using software. The N-terminal conservative sequence containing 309 amino acids was selected as the target peptide, and its corresponding gene sequence with modification based on prokaryotic cells-preferred codon was synthesized and inserted into plasmid pET28b(+). The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The expressed fusion protein was purified using Ni-affinity chromatography. The purified protein was injected along with adjuvant in rabbits to prepare the polyclonal antibodies against PPK.
Results And Conclusion:
PPK fusion protein expressed by E. coli was purified successfully using Ni-affinity chromatography. ELISA result demonstrated that the harvested rabbit anti-sera against PPK had a high titer of 1:512 000, and Western blotting showed a good specificity of the antibody, which can be used further study of the role of PPK in the pathogenesis of Proteus mirabilis infection.
Insights
Researchers successfully expressed and purified polyphosphate kinase (PPK) from Proteus mirabilis. They also developed a highly specific polyclonal antibody against PPK, valuable for future studies on Proteus mirabilis infections.
Area of Science:
- Microbiology
- Immunology
- Protein Biochemistry
Background:
- Polyphosphate kinase (PPK) plays a role in the pathogenesis of Proteus mirabilis.
- Understanding PPK function requires specific antibodies for detection and study.
Purpose of the Study:
- To express and purify polyphosphate kinase (PPK) from Proteus mirabilis.
- To generate a polyclonal antibody against PPK for further research.
Main Methods:
- PPK gene sequence was synthesized and cloned into a pET28b(+) plasmid.
- Recombinant protein was expressed in E. coli and purified using Ni-affinity chromatography.
- Polyclonal antibodies were prepared by immunizing rabbits with the purified PPK fusion protein.
Main Results:
- Purified PPK fusion protein was successfully obtained.
- ELISA confirmed a high titer (1:512,000) of anti-PPK antibodies in rabbit sera.
- Western blotting demonstrated the high specificity of the generated antibodies.
Conclusions:
- The study successfully produced a specific polyclonal antibody against Proteus mirabilis PPK.
- This antibody is suitable for further investigations into the role of PPK in Proteus mirabilis pathogenesis.