Krishnan Anuja, Malik Gunjan, Garg Lalit C
Department of Molecular Medicine, School of Interdisciplinary Science and Technology, Jamia Hamdard, New Delhi- 110062, India.
Gene Regulation Laboratory, National Institute of Immunology, New Delhi, 110067, India.
Protein Pept Lett. 2024;31(9):696-705. doi: 10.2174/0109298665325330240828115712.
To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of and B subunit of heat-labile enterotoxin (LTB) in the functional activity of L. donovani.
Visceral leishmaniasis, caused by the protozoan parasite , is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease (gp63) has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival.
The epitope corresponding to the catalytic motif of gp63 of was fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of promastigotes .
The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin (LTB) of , a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes .
It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein (LTB) could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.
研究抗重组嵌合体抗体对杜氏利什曼原虫功能活性的抑制潜力，该重组嵌合体由gp63的催化表位和不耐热肠毒素（LTB）的B亚基组成。
由原生动物寄生虫引起的内脏利什曼病是一个主要的健康问题，在热带地区可导致死亡。原生动物蛋白酶在该疾病的发病机制以及通过对抗宿主的固有免疫反应（即补体介导的裂解和吞噬作用）来建立感染过程中起着关键作用。据报道，一种表面结合的金属蛋白酶（gp63）是导致逃避补体介导的裂解、切割宿主细胞外和细胞内底物从而导致在吞噬溶酶体内存活的主要毒力因子。
将与杜氏利什曼原虫gp63催化基序相对应的表位与已知具有免疫原性的不耐热肠毒素的B亚基融合。将该嵌合体克隆到原核表达载体中，并使用镍-亚氨基三乙酸（Ni NTA）亲和层析进行纯化。制备针对纯化融合蛋白的抗体，并通过酶联免疫吸附测定（ELISA）分析其与gp63催化基序肽结合的能力。通过评估纯化的免疫球蛋白G（IgGs）对前鞭毛体蛋白酶活性和补体介导的裂解的影响，来评价融合蛋白抗体对gp63功能活性的作用。
本研究报告称，gp63催化表位与不耐热肠毒素（LTB）的B亚基的重组嵌合体，一种强大的体液反应佐剂，能够对催化表位产生显著的免疫反应。ELISA和蛋白质免疫印迹分析表明，抗融合蛋白抗血清能够识别天然的gp63。此外，它显著抑制了前鞭毛体的蛋白酶活性，并随后增加了补体介导的前鞭毛体裂解。
可以得出结论，含有杜氏利什曼原虫gp63蛋白催化基序和载体蛋白（LTB）的杂合蛋白能够引发可中和gp63功能活性的抗体，因此可能是亚单位利什曼病疫苗的潜在候选物。
