Small Interference RNA Knocks down the Expression of Gene CCP22

TGF β-Smad had wide signaling pathways involved in the regulation of various life processes, such as cell growth, differentiation, apoptosis, extracellular matrix formation and development. In these processes, Smad4 played a core role. Research shows tumorigenesis is associated with Smad4.

The domain (CCPmodule) of Complement control protein (CCP) was first discovered in 1987. There are two categories of proteins with CCP domain, regulators of complement activation (RCA) and non-regulators of complement activation (non - RCA). The CCP domain has an important function which can be combined with antigen molecules involved in cell signal recognition.

CCP22 is a new and not reported gene gained by using yeast two hybrid screening from DNA Library of human breast interacted with Smad4. The full-length of CCP22 gene is 4494bp, encoding a protein of 1497 amino acid residues. With existing data combined bioinformatics analysis, it is found that the protein encoded by this gene contains 21 CCP elements and 2.5 CCP sequence elements. So the gene was named CCP22.

Protein with different CCP domains, its domains are relatively conservative. But there are some differences in the number of them. Research shows that the proteins with CCP domains may also contain a transmembrane domain, receptor binding domain and other domains. Unlike other proteins with CCP domains, CCP22 only has a CCP domain. So in the CCP family of proteins, it is not clear about its ownership. The study on CCP22 contributes to the understanding of Smad4. It is possible to associate tumor development with immune complement, providing new molecular targets for drug therapy. To this end, small Interference RNA Knockdown is constructed. Western blot shows that siRNA can effectively reduce the expression of CCP22 protein.

At present, there are 3 main ways to get siRNA, chemical synthesis, in vitro transcription and siRNA recombinant expression vector construction. PSilencer2 is choosing as the siRNA expression vector. 1－U6－neo has RNApolIII promoter, and has the efficient, cheap and convenient advantages. The FlagCCP22 recombinant expression vector and CCP22siRNA recombinant vector are transfected into HEK293T cell line. Western blot shows that CCP22siRNA can effectively reduce the protein expression level of FlagCCP22. At the same time, CCP22siRNA can also reduce the endogenous CCP22 protein expression level, to lay a solid foundation for further study on the function of CCP22 protein.

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