콜레라 백신 후보 균주 개발 및 경구용 다가 백신 delivery system으로서의 이용에 관한 연구

초록

Purpose : To develop cholera vaccine candidate strains，we constructed Vibrio cholerae strains in which the expressed cholera toxin B subunit(CTB) is either secreted，or accumulated inside V. cholerae cells. Methods : For the construction of strain which accumulated CTB inside cells, pWM91-D3E4Kan was used for knock out of espE gene of type Ⅱ secretion pathway in classical strains, producing 0395-N1-E1 and 569B-N1-E1. The efficacy of O395-N1-E1 strain was evaluated in mice by determining the ability to induce anti-CTB IgG and vibriocidal activity. For the expression of ctxb gene in El Tor strain M7922，we used the mariner-based transposon(Tn) system. The engineered Tn，pSCGRl，was randomly transposed into the chromosome of M7922 to place the ctxb gene under the control of a strong promoter. The expression of the ctxb gene was screened by the ganglioside-dependent ELISA. Using pCP20 which encodes FLP- recombinase，chl marker of transposed cells was removed. Results : The constructed O395-N1-E1 strain accumulated 56% of expressed CTB inside cells. After immunization in mice，O395-Nl-El induced significantly high level of anti-CTB IgG response compared to the parental strain O395-N1. O395-N1-E1 also showed comparable vibriocidal activity as O395-Nl. Using pSCGRl in M7922，three CTB-secreting strains were selected after ELISA screening. The highest CTB-expressing isolate，M7922-C1 produced approximately 5.21ug/ml of CTB and was found to harbor an independent insertion in VC0972 gene which encodes a putative porin protein. Conclusions : The constructed V. cholerae strains may assist in preparing improved， effective and inexpensive cholera vaccine.

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