【作者】 许发芝；

【导师】 余为一；

【作者基本信息】 安徽农业大学 ， 微生物学， 2008， 博士

【摘要】 Ii链是一种非多态性的Ⅱ型跨膜蛋白，其中一个重要功能是确保新合成的MHCⅡ分子的胞内转运。MHCⅡ-Ii复合体的内体分选信号已被证明为Ii链胞浆尾部的两个独立的亮氨酸基元，即Leu⁷/Ile⁸和Pro¹⁵/Met¹⁶/Leu¹⁷。鸡Ii链胞浆尾部含有Leu⁸/Ile⁹和Val¹⁷/Leu¹⁸两个基元，但基元周围氨基酸是否对内体定位功能发挥作用还未见报道。我们利用大引物PCR定点突变法，将鸡Ii链胞浆尾部两个基元及其基元周围氨基酸分别突变为丙氨酸（Ala），然后连接到pEGFP-C1载体中，构建一系列突变体。利用脂质体Lipofectamine^TM2000介导的转染方法，将突变体转染到COS-7细胞，荧光显微镜下观察GFP-Ii融合蛋白的定位情况。结果显示，两个基元可以独立调控Ii分子的内化；突变Glu³、Glu⁴、Gln⁵、Ile⁹、Ser¹⁰、Ser¹⁵、Gly¹⁶、Val¹⁷或Pro¹⁹时，融合蛋白均定位于细胞浆，Ii分子内化功能丧失；而突变Arg⁶、Asp⁷、Ser¹¹、Asp¹²、G1y¹³或Ser¹⁴时，融合蛋白均定位于细胞内体，Ii分子内化功能保持。结果表明，鸡Ii链胞浆尾部两个亮氨基酸基元具有独立地内体定位信号功能，同时，两个基元功能的正确发挥需要周围氨基酸特定的空间结构支持。各类动物Ii链的跨膜区是进化中最保守的部分。研究发现，跨膜区在Ii分子自身聚合成三聚体中发挥重要的作用，在缺乏胞外区的情况下，跨膜区可以使Ii有效聚合成三聚体状态。已证实人Ii链跨膜区Gln⁴7，Thr⁴9和Thr⁵0三个氨基酸对MHCII-Ii复合物的形成是必须的。我们应用大引物PCR突变法，将鸡Ii基因的Gln⁴7和Thr⁵⁰氨基酸突变，构建了C1-Q47A和C1-T50A重组质粒。同时，MHCⅡ分子的α链和β链被亚克隆到pEGFP-N1载体，构建了GFP-α、GFP-β重组质粒。我们将C1-Q47A或C1-T50A和GFP-α、GFP-β共转染COS-7细胞，利用Western blot和免疫共沉淀对鸡Ii链跨膜的功能进行初步探讨。结果表明，突变Ii链跨膜区Gln⁴⁷或Thr⁵⁰氨基酸后，MHCⅡ-Ii复合物的形成受限。Ii链可以应用于构建Ii-抗原表位嵌合体。一些研究者用Ii链序列作为基本的框架，将CLIP区用CD4+T细胞抗原表位基因序列替代，将该嵌合基因插入真核表达载体，构建的嵌合体中的抗原表位能够有效地呈递给T细胞。关于鸡Ii分子CLIP替代的DNA疫苗目前没有研究报道，我们利用3轮重叠PCR将鸡Ii基因的CLIP区去除，此时恰好形成了一个HindⅢ酶切位点，再通过常规PCR扩增NDV的F343抗原表位基因，该基因片段长33个氨基酸，两段带有HindⅢ酶切位点。将F343基因通过单酶切连接入Ii分子内，构建了C1-Ii-F343内源性靶向抗原表位嵌合体DNA疫苗。同时，构建了C1-F343非靶向DNA疫苗作为对照。30只6-8周龄的雌性BALB/c小鼠随机分为5组，用内源性靶向DNA疫苗（C1-Ii-F343）和非靶向DNA疫苗（C1-F343）于股四头肌进行免疫,生理盐水、C1和C1-△CLIP作为研究对照。经过3次免疫后，取外周血对小鼠的体液免疫进行检测，免疫结果显示，只有C1-Ii-F343和C1-F343免疫组小鼠可以检测到针对F343抗原的特异性抗体，抗体滴度分别达到6400和1600，内源性靶向DNA疫苗组明显高于非靶向DNA疫苗组。构建抗原表位DNA替换鸡Ii分子的CLIP片段基因疫苗，为NDV的双靶向疫苗奠定实验基础。更多还原

【Abstract】 The invariant chain （Ii） is a non-polymorphic type ? transmembrane protein. A major function of Ii is to ensure the targeting of newly synthesized MHC ? to the endocytic pathway, the endosomal sorting signal for newly synthesizedαβIi complexes were identified as two independent motifs residues at positions Leu⁷/Ile⁸ and Met¹⁶ /Leu¹⁷ in the Ii cytoplasmic tail. Two similar signals, Leu⁸/Ile⁹ and Val¹⁷ /Leu¹⁸, were found in the cytoplasmic tail of chicken Ii. But the functional properties of the amino acids around the two motifs are not reported in chicken Ii so far. Two motifs and the amino acids around the motif residues were mutated to Ala by PCR-based megaprimer method for site-directed mutagenesis and ligated to the vector pEGFP-C1, the recombinant plasmid constructed respectively. These mutants were transiently transfected into the COS cells by Lipofectamine^TM2000, and then located fusion proteins were observed in the fluorescent microscope. Two Leu-based motifs both independently mediate efficient sorting to the endocytic pathway. When mutated Glu³, Glu⁴, Gln⁵, Iie⁹, Ser¹⁰, Ser¹⁵, Gly¹⁶ or Val¹⁷ to Ala, the GFP were detected on the plasma membrane, While Arg⁶, Asp⁷, Ser¹¹, Asp¹², Gly¹³ or Ser¹⁴ were mutated to Ala, GFP were located in the intracellular vesicles. Our results thus confirm that the cytosolic tail of chicken Ii comprises two independent endosomal sorting signals that function in internalization and a functional Leu-based sorting signal requires specific geometrically neighboring residues.The most conserved region of Ii is in the tranmembrane （TM） domain. It was reported that tranmembrane played an important role in Ii self-polymerizing to trimerization, even though absence cytoplasmic side of the membrane. It was confirmed that Gln⁴⁷, Thr⁴⁹ and Thr⁵⁰ in the tranmembrane were pre-requisite in the form of MHCII-Ii. Gln⁴⁷ and Thr⁵⁰ of chicken Ii were mutated to Ala by PCR-based megaprimer method for site-directed mutagenesis and ligated to the vector pEGFP-Cl, the recombinant plasmid C1-Q47A and C1-T50A constructed respectively. The recombinant vector N1-MHCII-αand N1-MHCII-βwere constructed by inserting PCR products into the plasmid pEGFP-Nl. C1-Q47A（C1-T50A） and N1-MHCII-α, N1-MHCII-βwere co-transfected into the COS-7 cell strain, using Western blot and Immunoprecipitation to examined the function of Ii tranmembrane.Ii was applied for constructing chimera gene containing antigen epitope. Ii as the basic framework would be used to substitute CLIP district for CD4+T cells epitope sequences, and the chimeric gene frangments were cloned into the eukaryotic expression vector. The construction of the chimera of epitope can be effectively presented to T cells. There is no research on the CLIP alternative DNA vaccine in chicken Ii, we used three overlapping PCR to remove the CLIP district of chicken Ii, at this time just formed a Hind? restriction site. And then by conventional PCR, NDV F343 epitope fragments were amplified with Hind? restriction sites, about 33 amino acids. F343 gene fragments were connected to the Ii by Hindlll and constructed the C1-Ii-F343 endogenous targeting epitopes chimeric gene vaccine. At the same time, construction of the C1-F343 gene vaccine is as for a non-targeting control. 30 6 to 8-week-old female BALB / C mice were divided into five groups targeted by endogenous gene vaccine （C1-Ii-F343） and non-targeting vaccine （C1-F343） in the quadriceps Immunization, saline, C1, and C1-△CLIP as a study control. After three times after immunization, blood from the humoral immunity in mice for testing, immunization results showed that only C1-Ii-F343 and C1-F343 immune mice can be detected for the F343-specific antigen antibody, antibody titers 6400 and 1600, respectively, within the target gene-derived vaccine was significantly higher than non-targeted vaccine group. Construction of epitope gene replacement chicken Ii CLIP fragment of the DNA vaccine was based on the two targeting experimental basis.更多还原