꼼치에서 특징적으로 발현되는 새로운 유전자 곰신의 분리 및 동정

Molecular Cloning and Identification of Novel Genes, Gomsin, Characteristically Expressed in Snailfish, Liparis tanakae

점액질이 풍부한 꼼치 조직에서 NIH 3T3 세포주를 이용하여 subtracted cDNA 라이브러리를 얻어 200례의 클론을 제작하였다. 이 클른 중에서 비반복성 유전자를 선택하고, RNA in situ hybridization을 실행하여 꼼치 조직에서 특이하게 발현되는 곰신 클론(C90-171)을 선택하였다. 이 클론은 사람의 타액선 조직에서도 특이하게 발현되는 유전자로서 이를 확인하기 위하여 C90-171(곰신) 항체를 제작하였다. 꼼치의 cDNA 라이브러리에서 곰신의 항체를 통하여 스크리닝한 결과 PRP(proline-rich protein)와 가장 많이 교차반응하며, 면역조직화학적 염색으로 PRP와 유사한 양성반응으로 나타나 PRP와 유사한 기능을 하는 단백질로 사료된다. 또한 타액 내에서의 꼼치 단백질의 분해에 대한 실험결과 거의 분해가 일어나지 않는 것으로 보아, 곰신은 꼼치의 몸통을 보호하는 유전물질일 뿐만 아니라, PRP와 유사하게 조직을 보호하는 안정된 새로운 기능성 단백질로 사료된다.

In order to obtain the specific genes of snailfish a subtracted cDNA library was constructed, and analysed by sequencing and GenBank search. Among them C90-171 clone was turned out to be genes showing low homology and nonredundant genes. This novel clone was named Gomsin(C90-171). Gomsin was shown to be intensely expressed in the epithelial cells, some mesenchymal cells, and sheaths of muscle bundles in the result of immunohistochemistry. In the cross reaction assay of Gomsin antibody against various human tissues, the Gomsin was strongly expressed in the ductal and acinar cells of salivary glands, which was similar to the expression patterns of proline-rich proteins(PRPs) of human. The antibody raised against the Gomsin was clearly cross-reacted with human salivary PRPs and also recombinant proteins of human PRPs in the Western blot and immunoprecipitation analysis. Contrast to the salivary PRPs, the Gomsin was not easily degraded in the mixed saliva, but rapidly attacked on the cultured keratocytes in vitro. The simulated protein structure of Gomsin was similar to the whorled pattern of PRPs, even though the amino acid sequence of Gomsin was quite different from those of PRPs. These data suggest that the Gomsin is a characteristic matrix protein in the skin and body of snailfish, which is also utilized for the tissue protection in the similar way to the PRPs of human muco-secretory organs.