간흡충 항원 유전자의 클로닝 및 재조합 항원을 이용한 혈청학적 진단

Abstract

[한글]

토끼의 담도로부터 얻은 살아 있는 간흡충을 protease inhibitor cocktail이 들어 있는 RPMI배지에 넣고 유지하여 분비배설항원을 얻었다. 이 분비배설항원 중 21 kDa 부위의 단백질을 면역시킨 흰쥐의 혈청과 간흡충증 환자 혈청으로 간흡충 성충의 cDNA library를 immunoscreening하여 강하게 반응하는 양성 clone을 선별해 내었다. 간흡충증 환자 혈청을 이용해 분리한 2개의 clone중 하나는 9개의 amino acids가 23회 반복되는 구조를 포함하는 265개의 amino acids로 구성되었고 glycine의 비중이 높아 GRCSP2라 명명하였다(GenBank Accession number AF461709). 다른 하나는 10개의 amino acids가 22회 반복되는 구조를 포함하는 269개의 amino acids로 구성되었고 proline의 비중이 높아 PRCSP라 명명하였다(GenBank Accession number AF461711). 간흡충 분비배설항원을 면역시킨 흰쥐의 혈청을 이용해 분리한 4개의 clone은 모두 흡충류의 myoglobin과 유사성이 높아 C. sinensis myoglobin이라 하였고 이는 150개의 amino acids로 구성되었다(GenBank Accession number AF461710). GRCSP2와 PRCSP는 간흡충 total RNA 상에서 모두 1.05 kb 크기로 발현되었고 C. sinensis myoglobin은 0.57 kb 크기로 발현되었다. Southern blotting 결과 3가지의 유전자 모두 간흡충 genomic DNA에 존재함을 확인하였다. GRCSP2와 PRCSP의 recombinant protein을 purification하여 간흡충증의 진단에의 민감도를 Western blotting으로 검사해 본 결과 각각 93.3%와 86.7%를 나타냈으며 폐흡충증, 요코가와흡충증, 회충증, 스파르가눔증 환자 혈청에 교차반응을 보였고, 특히 PRCSP가 더 많은 교차반응을 나타냄을 확인하였다.

[영문]

Clonorchis sinensis adult worms obtained from the biliary tract of an experimentally infected rabbit were suspended in RPMI including a protease inhibitor cocktail in order to obtain C. sinensis excretory-secretory(ES) antigens. A C. sinensis cDNA library was screened with immune rat sera which were immunized

with the 21 kDa protein of ES antigens and also screened with C. sinensis-infectedm human sera. Two clones were isolated by using C. sinensis-infected human sera. The cDNA insert of one clone contained a single open reading frame(ORF) of 795 base pairs encoding for 265 amino acids including 23 tandem repeats of 9 amino acids.

Because glycine was a major component in its content, the protein was named GRCSP2(GenBank Accession number AF 461709). The cDNA insert of the other contained a single ORF of 807 base pairs encoding for 269 amino acids including 22 tandem repeats of 10 amino acids. Because proline was a major component in its content, the protein was named PRCSP(GenBank Accession number AF 461711). Four clones were isolated by using the immune rat sera against 21 kDa protein of ES antigens. The cDNA inserts of four clones were very similar and contained a single ORF of 450

base pairs encoding 150 amino acids. Using BLAST program, we could find significant similarity between the cloned gene and a trematode myoglobin gene such as Paramphistomum epiclitum, so the protein was named C. sinensis myoglobin(GenBank Accession number AF 461710). Northern blotting was performed to examine the expression of the cloned gene in C. sinensis. When total RNA from C. sinensis was hybridized to the GRCSP2 gene, PRCSP gene, and C. sinensis myoglobin gene, a single band of 1.05 kb, 1.05 kb, and 0.57 kb was detected respectively. The results of Southern blot hybridization indicated that each cloned gene was present within the C. sinensis genome. Purified recombinant GRCSP2 and PRCSP showed high sensitivity for serodiagnosis, 93.3% and 86.7%, respectively. But the cross-reactivity of each recombinant protein was found agains