Recombinant Protein Expression and Antibody Production

The coding regions of hcp (VC1415) and hlyA (VCA0223) were amplified and cloned into the plasmids pET28a and pCold-TF (Takara), respectively. These recombinant plasmids were transferred into E. coli BL21(DE3) strain for protein expression. The His-tagged recombinant proteins were purified using Ni²⁺-NTA affinity chromatography, as was previously described.^{66 (link)} To prepare anti-Hcp and anti-HlyA antiserum for Western blotting analysis, 6-week-old Kunming female mice (Experimental Animal Center, Zunyi Medical University) were randomly assigned to each group (n = 10) and raised in a specific pathogen-free (SPF) environment. Mice were immunized subcutaneously with 30 µg of recombinant Hcp and HlyA proteins with an equal volume of aluminum adjuvant on days 1, 14, and 28. Blood samples were collected from the hearts on day 42, and the serum titers were tested by ELISA.

Free full text: Click here

Wang G., Fan C., Wang H., Jia C., Li X., Yang J., Zhang T., Gao S., Min X, & Huang J. (2022). Type VI secretion system-associated FHA domain protein TagH regulates the hemolytic activity and virulence of Vibrio cholerae. Gut Microbes, 14(1), 2055440.

Publication 2022

pET28a pCold-TF

Adjuvant Affinity chromatography Aluminum Antiserum Blood E coli Elisa Experimental animal Female Hearts Kunming mice Mice Plasmids Proteins Recombinant proteins Serum Specific pathogen free Strain Western blotting analysis

Corresponding Organization : Zunyi Medical University

Top 5 similar protocols

Variable analysis

independent variables

Recombinant Hcp and HlyA protein expression in E. coli BL21(DE3) strain

dependent variables

Purification of His-tagged recombinant Hcp and HlyA proteins using Ni2+-NTA affinity chromatography
Antibody production against Hcp and HlyA proteins in Kunming female mice

control variables

Use of pET28a and pCold-TF plasmids for protein expression
Immunization of Kunming female mice in a specific pathogen-free (SPF) environment
Use of aluminum adjuvant for immunization

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!