Deveropmental biotechnological analysis of host defense mechanisms against protozoan infection

(1)基因敲除小鼠实验采用巨噬细胞清道夫受体（SR）敲除小鼠研究宿主对原生动物感染的防御机制。由于已知SR可以结合非常广泛的配体，包括细菌病原体和介导巨噬细胞粘附，因此了解SR敲除小鼠对原生动物感染的反应将是有趣的。腹腔内接种微小巴贝虫后，每天通过计数寄生红细胞监测寄生虫血症。结果发现，SR-/-和SR+/+小鼠的寄生病程无明显差异，但感染后16-30 d， SR-/-小鼠的细胞体积减少程度低于SR+/+小鼠。SR-/-组小鼠感染微螺旋体后脾脏重量的增加也小于SR+/+组。流式细胞术研究显示，感染后SR-/-小鼠脾脏中thy1.2 +和CD4+ T细胞的百分比仍高于SR+/…More +小鼠。这些结果表明，SR敲除小鼠可能成为分析原生动物感染的有用模型，特别是在恢复过程方面，尽管它似乎没有直接参与对微孢子虫感染的防御机制。使用其他类型的敲除小鼠，如IFN γ和iNOS，也进行了研究。(2)产生携带原生动物genesag - 1 （p30）的转基因小鼠，该基因是弓形虫的主要表面蛋白，被认为是侵袭宿主细胞过程中的重要配体。如果宿主细胞产生p30蛋白并干扰寄生虫的蛋白，那么宿主就会对寄生虫的入侵产生抵抗力，或者由于缺乏对p30抗原的免疫反应，宿主更容易受到感染。为了产生携带p30基因的转基因小鼠，将含有p30 cDNA与CAG启动子融合的3.3kb DNA片段微注射到C57BL/6J、BALB/c或F1 （C57BL/6J x C3H/He）小鼠单细胞胚胎的其中一个原核中，该片段已被证明可指导转基因小鼠中普遍存在的报告基因的表达。将胚胎移植到假妊娠ICR小鼠的输卵管中，在第一次使用近交系的实验中，从159只小鼠中获得了2只p30阳性的建立者（雄性C57BL/6J和雌性BALB/c）。此外，我们还利用F1 （C57BL/6J x C3H/He）胚胎获得了几只表达该基因产物的P30-founder小鼠，并将该基因传递给下一代。据我们所知，这是第一个携带弓形虫原生动物基因的转基因小鼠。少

(l)Studies on gene knock-out miceExperiments on the host defense mechanisms against protozoan infection were performed using a macrophage scavenger receptor (SR) knock-out mice. As the SR is known to bind an extraordinary wide range of ligands including bacterial pathogens and mediate macrophage adhesion, it would be of interest to know the response of SR knock-out mice against protozoan infection. After intraperitoneal inoculation with Babesia microti, parasitemia was monitored daily by counting parasitized erythrocytes.It was found that the course of parasitemia was not different between SR-/- and SR+/+ mice but the decrease in packed cell volume at 16-30 days after infection was less obvious in SR-/- than in SR+/+ mice. Increase in spleen weight after B.microti infection was also smaller in SR-/- than in SR+/+ mice. Flow cytometric studies revealed that the percentages of Thy 1.2+ as well as CD4+ T cells in spleen remained relatively higher in SR-/- mice as compared to those in SR+/ … More + mice after infection. These results suggest that the SR knock-out mouse may become a useful model for the analysis of protozoan infection, especially with regard to the recovery processes, although it seems not to be directly involved in the defense mechanisms against B.microti infection.Studies using other types of knock-out mice, such as IFN gamma and iNOS, were also conducted.(2) Production of transgenic mice carrying protozoan geneSAG-i (p30), the major surface protein of Toxoplasma gondii, is considered as an important ligand in the process of host cell invasion. If this protein is produced by host cells and interfere with that of parasites, then the host will become resistant to parasite invasion, Or, it may become more susceptible to infection due to the lack of immune response against p30 antigen. To generate transgenic mice carrying p30 gene, a 3.3kb DNA fragments, containing p30 cDNA fused with CAG promoter, that has been shown to direct a ubiquitous expression of a reporter gene in transgenic mice, were microinjected into one of the pronuclei of one-cell embryos of C57BL/6J, BALB/c or F1 (C57BL/6J x C3H/He) mice. The embryos were transferred to the oviducts of pseudopregnant ICR mice, In the first series of experiment using inbred strains, two p30-positive founders (male C57BL/6J and female BALB/c) were obtained among 159 mice that developed from the injected eggs. Furthermore, we have obtained several P30-founder mice expressing the gene product and transmitting the gene to the next generation, by using F1 (C57BL/6J x C3H/He) embryos. As far as we are aware, this is the first transgenic mice bearing a protozoan gene derived from T.gondii. Less