PASSIVE-IMMUNITY TO YERSINIAE MEDIATED BY ANTI-RECOMBINANT V-ANTIGEN AND PROTEIN A-V-ANTIGEN FUSION PEPTIDE
PASSIVE-IMMUNITY TO YERSINIAE MEDIATED BY ANTI-RECOMBINANT V-ANTIGEN AND PROTEIN A-V-ANTIGEN FUSION PEPTIDE
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DOI：
10.1128/iai.62.10.4192-4201.1994
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发表时间：
1994-10-01
影响因子：
3.1
通讯作者：
BRUBAKER, RR
中科院分区：
文献类型：
--
作者：
MOTIN, VL;NAKAJIMA, R;BRUBAKER, RR
LcrV（V抗原），一种已知的不稳定的37.3-kDa单体肽编码的ca。鼠疫耶尔森氏菌、假结核耶尔森氏菌和小肠结肠炎耶尔森氏菌的70-kb Lcr质粒被认为是低钙应答的调节因子、毒力因子和保护性抗原。在本研究中，Y.鼠疫杆菌的基因克隆到蛋白酶缺陷型大肠杆菌BL 21中。所得重组V抗原在纯化过程中从C-末端发生显著降解，产生36、35、34和32至29 kDa的主要肽。针对这种裂解产物混合物的兔丙种球蛋白对10个最小致死剂量的Y提供了显着的保护。鼠疫菌（P < 0.01）和耶尔森氏菌（P < 0.01）。假结核（P < 0.02）。为了稳定V抗原并促进其纯化，构建质粒pPAV 13以编码lcrV和蛋白A的结构基因的融合体（即，除了V抗原的前67个N-末端氨基酸加上信号序列和免疫球蛋白G-结合结构域，但不是蛋白A的细胞壁相关区域）。所得到的融合肽，称为PAV，可以纯化的同质性在一个步骤中通过免疫球蛋白G亲和层析，并在此后是稳定的。兔抗PAV多克隆丙种球蛋白对10个最小致死剂量的Y.鼠疫菌（P < 0.005）和Y.假结核（P < 0.005），对Y.小肠结肠炎。在用过量PAV、克隆的完整V抗原或后者的大（31.5-kDa）截短衍生物吸收后保护失败，但在用较小（19.3-kDa）截短变体类似吸收后保留（P < 0.005），表明至少一个保护性表位存在于氨基酸168和275之间的内部。
LcrV (V antigen), a known unstable 37.3-kDa monomeric peptide encoded on the ca. 70-kb Lcr plasmid of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica, has been implicated as a regulator of the low-calcium response, virulence factor, and protective antigen. In this study, lcrV of Y. pestis was cloned into protease-deficient Escherichia coli BL21. The resulting recombinant V antigen underwent marked degradation from the C-terminal end during purification, yielding major peptides of 36, 35, 34, and 32 to 29 kDa. Rabbit gamma globulin raised against this mixture of cleavage products provided significant protection against 10 minimum lethal doses of Y. pestis (P < 0.01) and Y. pseudotuberculosis (P < 0.02). To both stabilize V antigen and facilitate its purification, plasmid pPAV13 was constructed so as to encode a fusion of lcrV and the structural gene for protein A (i.e., all but the first 67 N-terminal amino acids of V antigen plus the signal sequence and immunoglobulin G-binding domains but not the cell wall-associated region of protein A). The resulting fusion peptide, termed PAV, could be purified to homogeneity in one step by immunoglobulin G affinity chromatography and was stable thereafter. Rabbit polyclonal gamma globulin directed against PAV provided excellent passive immunity against 10 minimum lethal doses of Y. pestis (P < 0.005) and Y. pseudotuberculosis (P < 0.005) but was ineffective against Y. enterocolitica. Protection failed after absorption with excess PAV, cloned whole V antigen, or a large (31.5-kDa) truncated derivative of the latter but was retained (P < 0.005) upon similar absorption with a smaller (19.3-kDa) truncated variant, indicating that at least one protective epitope resides internally between amino acids 168 and 275.