Analysis on protective antigen of Babesia parasites by developmental engineering

1. 小巴贝斯虫在小鼠中产生自限性感染，恢复后的小鼠对再次感染具有抵抗力。本研究以巨噬细胞清道夫受体（SR）、诱导型一氧化氮合酶（iNOS）和γ干扰素（IFN-γ）敲除小鼠为实验对象，探讨了小鼠对微芽胞杆菌的保护机制。与SR+/+小鼠相比，SR-/-小鼠堆积细胞体积的减少和脾脏重量的增加不明显。在感染的早期阶段，inos缺陷小鼠的寄生虫血症比对照小鼠增加得更快，尽管它们从感染中恢复过来。IFN-γ敲除小鼠不能控制原发感染，也不能预防侵袭性感染。在大肠杆菌和重组杆状病毒感染的昆虫细胞中表达了罗德海贝氏杆菌抗原p26。用重组抗原和Freund佐剂免疫BALB/c小鼠，对攻毒感染的存活率为40- 100%。MAb 1-5H可识别微氏B.microti的58 kda蛋白，并与b.m rohaini的60 kda蛋白发生交叉反应。编码p58蛋白的完整核苷酸序列包含1629个碱基对（bp）的开放阅读框，包含542个氨基酸残基，被命名为p58基因。蛋白同源性研究表明，该蛋白与伴侣蛋白的η亚基具有显著的氨基酸同源性。BEG3单抗识别马b.a i的19kDa抗原，并在体外抑制马b.a i的增殖。单克隆抗体BCllD识别出B.caballi裂殖子中一个48kda的蛋白构象蛋白。克隆了一段长达50kDa的gibsoni蛋白cDNA，并确定了其完整的核苷酸序列。马布氏杆菌（MEA-1）、caballi布氏杆菌（BC48）和gibsoni布氏杆菌（p50）的重组蛋白可作为ELISA或胶乳凝集试验等血清诊断方法的抗原。

1. Babesia microti produces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the protective mechanisms against B.microti were examined using macrophage scavenger receptor (SR), inducible nitric oxide synthase (iNOS), and gamma interferon (IFN-γ) knock-out mice. Decrease of packed cell volume and increase of spleen weight were less obvious in SR-/- mice than in SR+/+ mice. Parasitemia in iNOS-deficient mice increased more rapidly than those of control mice during the early stage of infection, although they recovered from the infection. IFN-γ knock-out mice could not control primary infection, nor protect against challenge infection.2. B.rodhaini antigen p26 was expressed in Escherichia coli and in insect cells infected with a recombinant baculovirus. BALB/c mice immunized with both recombinant antigens and Freund's adjuvants showed 40- 100% survival rate against challenge infection. MAb 1-5H recognized a 58-kDa protein of B.microti and was found to cross-react with a 60-kDa protein of B.rodhaini. The complete nucleotide sequence encoding p58 protein contained an open reading frame of 1,629-base pair (bp) nucleotides that consists of 542 amino acid residues and the gene was named as p58 gene. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin.3. MAb BEG3 recognized 19kDa antigen of B.equi and inhibited the multiplication of B.equi in vitro. MAb BCllD recognized a 48-kDa protein rhoptry protein of B.caballi merozoite. A cDNA encoding a 50kDa protein of B.gibsoni was cloned and the complete nucleotide sequence was determined. Recombinant proteins of B.equi (MEA-1), B.caballi (BC48) and B.gibsoni(p50) were found to be useful as antigens for serodiagnostic methods such as ELISA or latex agglutination test.