1.Basic Information:
CldN11 (Claudin 11) is an intercellular connexin that functions primarily in the central nervous system. It is expressed in myelin cells and is involved in maintaining the structure and function of the myelin sheath of nerve fibers. In order to gain a better understanding of the function of CLDN11 and its relationship with neurological diseases, we designed the following experimental protocol.
2.Gene cloning:
First, total RNA is extracted from human brain tissue and the mRNA is transcribed into cDNA using reverse transcriptase. Then, specific primers were designed to amplify the full length of the ClDN11 gene in the PCR reaction. The amplified product is enzymatically digested with the appropriate plasmid vector and ligated to construct a recombinant plasmid.
Catalog No. PA1000-5546
3.Transforming E. coli:
Transform the recombinant plasmid into E. coli and screen positive colonies on LB agar plates. Identification of the correct recombinant plasmid by PCR and restriction digestion.
4.Expression of recombinant proteins:
Single clones were picked from positive colonies and pre-incubated in LB medium overnight on a 37-shaker bed. Take one of the pre-culture strains and put them into the medium containing an appropriate amount of LB AMP (containing an appropriate amount of antibiotics), and re-incubate until the OD600 reaches 06-0.8。Add IPTG (isopropyl--D-thiogalactoside) to a final concentration of 05 mm, continue to incubate at low temperature (e.g., 16) for 12-16 h to induce overexpression of CLDN11 recombinant protein.
5.Purification of recombinant proteins:
After IPTG induction, the bacterial pellet is collected and the cell pellet is suspended with wash buffer. The cells are disrupted using ultrasonic disruption techniques to release cellular proteins including clDN11. Remove cell membranes and broken cells by centrifugation, and purify the supernatant with an affinity chromatography column such as his-tag affinity resin to remove impurities. Recombinant proteins are eluted and protein concentrations are determined using protein detection methods such as BCA.
6.Functional Assessment:
The expression of ClDN11 protein on the intercellular membrane was detected by transfection of human myelin cells (such as Olig2 cell line) or rat myelin cell cell line, and immunocytochemistry or co-immunoprecipitation. Furthermore, the effect of CLDN11 on myelin structure and function of myelin sheath can be evaluated by myelination assay, cell adhesion assay, conduction velocity assay and other methods of transfected cells.
Through the above experimental design, we will be able to obtain the purified CLDN11 recombinant protein and conduct a preliminary evaluation of its function, further revealing its important role in the nervous system and its association with neurological diseases.