Leishmania major: Molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences

被引:34
作者
ReyLadino, JA
Joshi, PB
Singh, B
Gupta, R
Reiner, NE
机构
[1] UNIV BRITISH COLUMBIA, DEPT MED, DIV INFECT DIS, FAC MED, VANCOUVER, BC V5Z 3J5, CANADA
[2] UNIV BRITISH COLUMBIA, DEPT MICROBIOL, FAC MED, VANCOUVER, BC V5Z 3J5, CANADA
[3] UNIV BRITISH COLUMBIA, DEPT IMMUNOL, FAC MED, VANCOUVER, BC V5Z 3J5, CANADA
[4] UNIV BRITISH COLUMBIA, DEPT IMMUNOL, FAC SCI, VANCOUVER, BC V5Z 3J5, CANADA
[5] UNIV BRITISH COLUMBIA, DEPT MICROBIOL, FAC SCI, VANCOUVER, BC V5Z 3J5, CANADA
[6] UNIV BRITISH COLUMBIA, DEPT MED, FAC SCI, VANCOUVER, BC V5Z 3J5, CANADA
[7] UNIV BRITISH COLUMBIA, FAC MED, DEPT MED GENET, VANCOUVER, BC V6T 1Z3, CANADA
[8] UNIV BRITISH COLUMBIA, FAC SCI, DEPT MED GENET, VANCOUVER, BC V6T 1Z3, CANADA
[9] MCMASTER UNIV, HLTH SCI CTR, DEPT BIOCHEM, HAMILTON, ON L8N 3Z5, CANADA
[10] VANCOUVER HOSP & HLTH SCI CTR, RES INST, VANCOUVER, BC V5Z 3J5, CANADA
基金
英国医学研究理事会;
关键词
Leishmania major; protozoa;
D O I
10.1006/expr.1996.4137
中图分类号
R38 [医学寄生虫学]; Q [生物科学];
学科分类号
07 ; 0710 ; 09 ; 100103 ;
摘要
Heat shock proteins (HSP) in the size range of M(r) 60,000 are major targets of the immune response in vivo. The leishmania heat-inducible proteins of M(r) 65-67,000 are expressed at relatively high levels in infected macrophages (Infection and immunity 1993, 61, 3265-3272) and may be important targets of the host response. To facilitate further studies concerned with these proteins, the HSP60 gene of Leishmania major was cloned, sequenced, and expressed. A lambda EMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was characterized. Sequence analysis revealed an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M(r) 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from Trypanosoma cruzi. Less extensive amino acid sequence similarity (48%) was observed between that leishmania HSP60 and the corresponding human protein. Notably, significant regions of sequence dissimilarity between the leishmania and human proteins were identified principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was subcloned into the pET-3a vector and expressed in Escherichia coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from Colombia for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. This recognition had specificity in that these same sera showed little or no reactivity with either recombinant mycobacterial HSP65 or recombinant human HSP60. These findings indicate that patients with tegumentary forms of leishmaniasis have humoral responses to leishmania HSP60. Further studies of this protein will clarify its importance as a target of the immune response and as a potential antigen for serodiagnosis. (C) 1997 Academic Press.
引用
收藏
页码:249 / 263
页数:15
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