1. Basic information analysis:
Polymeric immunoglobulin receptor (PIGR) protein is a transmembrane glycoprotein whose main function is to mediate immunoglobulin A (IgA) transport in mucosal immunity. PIGR is mainly found in a variety of tissues such as the immune system. The structure of the protein consists of an extracellular part in which the extracellular part binds to IgA and facilitates its transport in the mucosa. Catalog No. PA1000-6941
2. Experimental design:
1.Cloning the PIGR gene: RNA is extracted from human tissues (e.g., lungs, small intestine, etc.) for reverse transcription and amplification of the PIGR gene using polymerase chain reaction (PCR).
2.Construct an expression vector: Ligation of the amplified PIGR gene fragment with an appropriate expression vector (e.g., PET-32A) to generate a eukaryotic expression plasmid of the PIGR recombinant protein.
3.Transfection of host cells: The constructed PIGR recombinant plasmid was transfected into an adapted host cell (e.g., mammalian cell line HEK-293T) and expressed PIGR protein using cell transfection technology.
4.Screening of stable cell lines: Stable cell lines with high expression of PIGR protein were screened through screening and dilution.
5.Purification of expressed proteins: Purification of pigr proteins is achieved by using affinity purification techniques, such as the Ni-NTA affinity column purification method, which uses the tag portion of the pigr protein with histidine residues to bind to nickel ions on the column.
6.Protein identification: The molecular weight and immune specificity of purified PIGR protein were identified by sodium eicosanylsulfonate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.
7.Functional analysis: The functionality of purified PIGR proteins is verified by performing IgA binding experiments (e.g., ELISA) or transport model experiments (e.g., cell and mouse models).
Through the above experimental design and analysis, we can achieve the following goals:
1.The pigr gene was cloned and expressed to obtain a cell line stably expressing the protein.
2.Purify PIGR proteins and determine their molecular weight and immune specificity.
3.The binding ability of PIGR protein to IgA was verified and its functionality was evaluated.
The final experimental results will provide basic data for the application of PIGR recombinant protein in mucosal immunity research and related diseases**, and lay a foundation for further research on the function and mechanism of the protein.