한국 여성의 Lactadherin 유전자 Cloning과 발현 연구
Cloning and Expression of Lactadherin Gene from Korean Women
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Lactadherin은 모유의 유지방구막의 당단백질의 하나로 mucin, butyrophilin과 결합된 복합체이다. 특히 모유중의 mucin과 lactadherin은 출생 직후 면역력이 약한 유아를 병균의 침입으로부터 효율적으로 방어하여 초기 유아의 생존과 성장 및 발달에 매우 중요한 역할을 수행한다. Lactadherin은 유아 설사의 원인이 되는 rotavirus의 번식과 성장을 억제한다. 아울러 이 단백질은 새로운 혈관의 형성을 촉진하는 주요한 단백질로 알려져 있으며 이 단백질의 결핍이 치매의 발생과 관련되는 것으로 보고되고 있다. 본 연구는 이처럼 중요성이 강조되는 lactadherin에 대한 생화학적 및 생리학적인 연구를 하기 위한 기초연구를 진행하였다. 한국 여성의 유선조직에서 mRNA를 분리하였고 1.2kb lactadherin cDNA 유전자를 cloning하여 염기서열과 아미노산 배열을 결정하였다. 이 cDNA를 pET vector에 삽입하여 E. coli에서 43kD 단백질을 발현시켰으며 Western blot으로 확인하였으며, 이 단백질을 정제하여 토끼에서 항체를 생산하여, 한국 여성의 모유에서 발현되는 70, 55, 46, 30 kD의 band를 확인하였다. 아울러 백인 여성의 lactadherin 유전자와 한국 여성의 정상 및 유방암 조직의 유전자 비교에서 다양한 SNP가 관찰되었고 변이의 다형성이 관찰되었다.
Abstract
Lactadherin is a glycoprotein of human milk fat globule membrane that binds to mucin and butyrophilin forming the protein complex. Especially, mucin and lactadherin in human milk efficiently protect infants with poor immune functions right after birth from infections by microorganisms and play important roles for their early survival, growth and development. Lactadherin inhibits the propagation and growth of rotavirus that is a global pathogen causing infants' diarrhea. Recently this protein was known to promote neovascularization and its deficiency related to develop alzheimer's disease. In this study, the basic biochemical and physiological aspects of lactadherin were investigated. Messenger RNAs were isolated from mammary tissues from Korean women patients to clone a 1.2 kb cDNA and sequenced its DNA to determine its amino acid sequences. The cDNA was cloned to express its 43kD protein, which was confirmed by Western blot. The recombinant protein was purified and injected to 2 rabbits to raise antibodies against it. The semi-purified milk fat globule membrane proteins from Korean women was analyzed by Western blot using the rabbit antibody to give 70, 55, 46, 30 kD bands. Also several polymorphism and SNPs of lactadherin gene from Korean women were observed compared with those of Caucasian women.
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