Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
Biomed Environ Sci. 2010 Oct;23(5):333-40. doi: 10.1016/S0895-3988(10)60073-6.
LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.
A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.
Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.
The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.
LcrV 是抗鼠疫亚单位疫苗研发的重要组成部分。为降低 LcrV 的免疫抑制活性，本研究采用不同方法制备缺失 271 至 326 位氨基酸的重组 LcrV 变异体（rV270）。
设计了一种新策略，通过将 rV270-凝血酶-六组氨酸融合基因插入载体 pET24a 中，或插入六组氨酸-肠激酶-rV270 或六组氨酸-因子 Xa-rV270 融合基因插入载体 pET32a 中，产生非标记或天然 rV270 蛋白。经 Co(2+)亲和层析后，通过柱上切割 His 标签，再经 Sephacryl S-200HR 柱过滤层析，开发了一种纯化策略。
凝血酶、肠激酶和因子 Xa 去除 His 标签的收率分别为 99.5%、32.4%和 15.3%。经 Sephacryl S-200HR 柱过滤层析后，rV270 蛋白的纯度超过 97%。纯化的 rV270 吸附于磷酸盐缓冲液（PBS）中的 25%（v/v）Al(OH)₃佐剂，可诱导 BALB/c 小鼠产生针对 rV270 的高滴度抗体，并使其（100%存活）免受 10⁶ CFU 鼠疫强毒株 141 的皮下攻击。
用肠激酶或因子 Xa 可以制备完全天然的 rV270 蛋白，但它们对相应识别位点的切割活性极低。凝血酶切割是制备非标记 rV270 蛋白的有效策略，由于其成本相对较低且切割效率较高，因此易于大规模操作。重组 rV270 可作为鼠疫亚单位疫苗研发的关键成分。
