异亮氨酸拉链结构对可溶性CD40L生物学活性的影响

CD40配基(CD40L)为TNF超家族中的一员，瞬时表达于激活的CD4^ 的T淋巴细胞上．自然状态下膜上三聚体的CD40L通过和CD40受体(CD40R)结合，在免疫监视和肿瘤免疫中发挥作用．以PCR方法从peDNA3-CD40L质粒上扩增得到编码CD40L基因的胞外部分sCD40L，并将该片段插入大肠杆菌表达质粒pET30a上，构建得到大肠杆菌表达质粒pET30a-sCD40L；同时，在sCD40L的N端融合了异亮氨酸拉链(Isoleucine Zipper，IZ)结构，构建得到表达质粒pET30a-IZ-sCD40L．表达质粒在大肠杆菌中表达，得到可溶性的重组人CD40L和IZ-CD40L．SDS-PAGE分析结果表明重组人rhsCD40L和IZ-rhsCD40L在相应分子质量位置都有明显的条带产生，并得到Western blotting的确认．非SDS-PAGE电泳结果显示，异亮氨酸拉链结构有利于rhsCD40L蛋白形成聚合体结构．B细胞增殖实验和人骨髓瘤细胞株XG2凋亡实验结果都表明，异亮氨酸拉链结构提高了rhsCD40L的生物学活性．     

人扁桃体淋巴细胞cDNA质粒重组体的构建

目的：通过构建人扁桃体淋巴细胞cDNA质粒重组体，为深入研究淋巴细胞的生物学功能奠定实验基础。方法：采用DNA重组技术，以人新鲜扁桃体淋巴细胞为实验材料从中提取mRNA，以mRNA为模板合成cDNA，然后与质粒pSPORTI进行定向连接并转入到E．coliJM109中进行分子克隆。结果：阳性克隆占90％，阴性克隆占10％；转化率为1．35×104克隆／微开连接体系；插入片段长度为0．5～2．3Kb，平均1．3Kb。结论：人扁桃体淋巴细胞cDNA质粒重组体构建成功。     

人共刺激分子B7-1/CD80胞外编码区cDNA的克隆及序列分析

用RT－PCR法从人外周血单核细胞克隆了编码共刺激分子B7－1／CD80胞外区的cDNA，并用Snager链终止法进行测序，结果表明，所cDNA片段的序列与GenBank中已报道的人B7－1／CD80 cDNA序列的对应部分仅有两个碱基存在差异（658位A→T，773位A→G），这为探讨B7－1／CD80分子的生物学功能奠定了基础。     

丙型肝炎病毒NS3基因真核质粒的构建及其在人肝细胞中的诱导表达

目的：进行丙型肝炎病毒非结构蛋白3基因真核表达载体的构建,并分析其在体外培养的人肝细胞中的表达。方法：从含有丙肝病毒全长基因的重组质粒pBRTM／HCV1-3011表达载体中PCR扩增出HCVNS3基因片段,将其与表达载体pcDNA3．1（-）重组,得到重组的真核表达载体pcDNA3．1（-）／NS3。然后采用阳离子多聚体将其转染人肝细胞QSG7701,以免疫组织化学SP法及Western Blotting检测HCVNS3蛋白的表达。结果：所得到的NS3片段正确,序列正确,所构建的真核质粒成功转染QSG7701细胞并表达蛋白,表达的NS3蛋白相对分子质量为70000。结论：成功构建了丙型肝炎病毒非结构蛋白3基因的真核表达载体pcDNA3．1（-）／NS3,并且该载体在体外培养的人肝细胞中能有效表达特异性HCVNS3蛋白。     

人细胞周期蛋白cyclin E基因的克隆与真核表达载体的构建

目的:为研究细胞周期蛋白在肿瘤形成过程的分子机制,构建带FLAG标签的细胞周期蛋白E的真核表达载体,并检测其在瞬时转染HeLa细胞株中的蛋白表达。方法:通过RT-PCR扩增cyclinE基因编码cDNA,并将扩增的cDNA片段插入p3XFLAG-CMVTM-14真核表达载体,重组子经酶切分析和测序鉴定后,用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染HeLa细胞,用Western-blot技术检测其在HeLa细胞中融合蛋白的表达。结果:经酶切鉴定和测序分析证实人cyclin E的真核表达载体构建成功,并能在瞬时转染的HeLa细胞中表达。结论:成功构建了人cyclin E的真核表达载体,为进一步研究细胞周期蛋白的功能奠定了基础。     

HPV16 E6基因真核表达载体的构建

本研究目的是构建HPV16 E6真核表达载体,为细胞转染研究E6蛋白和p53Arg或p53Pro蛋白的相互作用以及作用后对细胞功能的影响奠定基础。应用PCR技术从质粒pSP64-HPV16 E6上扩增出HPV16 E6片断。根据Kozak序列对真核蛋白表达的影响,设计2条引物扩增出2个HPV16 E6目的片断均插入真核表达载体pcDNA3．1／myc-His（-）A中,构建pcDNA3．1／myc-His（-）A．HPV16 E6重组质粒。研究结果显示：经酶切和测序鉴定克隆的基因片段为HPV16 E6 cDNA;建立了pcDNA3．1／myc-His（-）A-HPV16 E6重组质粒。这表明,已成功构建HPV16 E6真核表达载体pcDNA3．1／myc-His（-）A-HPV16 E6。     

硫丹对长爪沙鼠血细胞免疫相关因子表达的影响

目的 探讨有机氯农药硫丹对长爪沙鼠血细胞免疫功能相关因子的影响。方法将50只长爪沙鼠随机分成5组,各组动物所用硫丹灌胃的剂量依次为：0mg／（kg·d）、0．4mg／（kg·d）、0．8mg／（kg·d）、1．6mg／（kg·d）和6．4mg／／（kg·d）。灌胃21d后,取血检测血细胞免疫功能相关因子的表达。结果药物处理后,红细胞表面CD59因子和B淋巴细胞表面CD35因子的表达量在各实验组和对照组间没有明显差异（P〉0．05）。红细胞膜表面CD58因子随硫丹浓度的升高显著下降,0．8mg,／（kg·d）、1．6mg／（kg·d）和6．4／mg／（kg·d）组较对照组有显著降低（P〈0．05）。结论硫丹能抑制长爪沙鼠红细胞膜表面CD58因子表达,但对红细胞膜表面CD59和B细胞表面的CD35的表达没有影响。     

HAb18G/CD147真核荧光蛋白的表达及鉴定

构建肝癌相关抗原HAb18G／CD147全长及缺失片段E51（第22—50位氨基酸缺失）的真核荧光蛋白表达载体,进一步研究该基因在肝细胞肝癌发生中的作用。通过PCR及Overlapping PCR的方法获得目的基因．与荧光载体pEGFP—N1双酶切、连接、转化E．coli JM109．构建含有肝癌相关抗原HAb18G／CD147全长及E51的真核荧光蛋白表达载体．并经限制性酶切及序列分析证明插入是否正确。用阳离子脂质体介导转染COS-7细胞,进行瞬时表达;荧光显微镜观察EGFP表达,通过流式细胞术检测蛋白的表达情况,明胶酶谱法鉴定其功能。成功地构建了真核荧光蛋白表达载体pEGFP—N1／HAb18G、pEGFP—N1/E51,并经限制性酶切及序列分析证明外源基因插入正确;流式细胞术鉴定该蛋白表达正确;功能实验证实缺失片段E51不具有诱导成纤维细胞分泌MMPs的功能．结果显示HAb18G／CD147基因第22—50位氨基酸与其刺激成纤细胞分泌MMP和功能有密切关系。实验结果为HAb18G蛋白分子的生物学功能研究奠定了基础。     

DNA免疫制备抗人CD154单克隆抗体及其轻链可变区基因序列分析

采用pcDNA3 1 hCD15 4重组质粒DNA免疫BALB c小鼠 ,制备抗人CD15 4单克隆抗体 ,得到了能分泌针对人T细胞表面CD15 4分子的特异性单抗的杂交瘤细胞株 (7E8)。通过RT_PCR扩增 7E8单抗轻链可变区基因 ,并对其进行克隆、测序、DNA序列分析和氨基酸推导 ,结果显示轻链可变区基因和氨基酸序列完全符合小鼠Ig轻链的结构特征 ,为制备基因工程抗体并应用于临床打下了基础。     

CD226分子siRNA慢病毒载体的构建和鉴定

为设计筛选针对人CD226分子的siRNA序列,构建相应的siRNA慢病毒载体,检测其对Jurkat/E6细胞膜表面天然CD226分子表达水平的影响,首先设计并合成4对siRNA双链寡聚核苷酸,与CD226分子真核表达载体共同转染293T细胞,挑选出最有效抑制CD226表达的序列。应用基因工程技术,将该序列连接于慢病毒载体pLKO.1中,构建携带针对目的基因CD226的siRNA慢病毒载体。使用慢病毒包装质粒混合物和构建好的慢病毒载体共转染293T细胞,收集上清,感染Jurkat/E6细胞系,对病毒感染效果进行检测。结果在4条针对CD226分子设计的siRNA序列中,siRNA—513最为有效的抑制了外源性CD226分子的表达,带有该序列的慢病毒载体pLKO—CD226可以完全抑制Jurkat/E6细胞上天然分子的表达。说明应用基因工程技术成功构建了针对CD226分子的RNA干扰慢病毒载体,为深入研究人类黏附分子CD226在机体免疫功能方面提供了技术支持。     

CD21 is a ligand for CD23 and regulates IgE production.

The molecule CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a type II transmembrane molecule expressed on many haemopoietic cell types. CD23 has pleiotropic roles in the control of lymphocyte behaviour, suggesting that CD23 may interact with another ligand in addition to IgE. To identify such a CD23 ligand, we expressed and purified full-length recombinant CD23, incorporated it into fluorescent liposomes and used these as a probe. We report here that fluorescent liposomes carrying CD23 interact specifically with the cell-surface protein CD21, identified as the receptor for Epstein-Barr virus and the complement receptor-2 on B cells, some T cells and follicular dendritic cells. In addition, fluorescent CD23-liposomes were shown to bind to hamster kidney cells (BHK-21) transfected with CD21 complementary DNA. The interaction between fluorescent CD23-liposomes and B cells or CD21-transfected BHK-21 cells was specifically inhibited by anti-CD21 and anti-CD23 monoclonal antibodies. Western blotting analysis revealed that 14C-labelled liposomes carrying CD23, in contrast to anti-CD21 antibodies, reacted with a subtype of CD21 molecules. Triggering of CD21 either with an anti-CD21 antibody or with recombinant soluble CD23 was shown to increase specifically interleukin-4-induced IgE production from blood mononuclear cells. These results demonstrate that the cell-surface protein CD21 is a ligand for CD23 and that the pairing of these molecules may participate in the control of IgE production.     

Enhancement of T cell immune response to lymphoma cells by CD28/CD80 alternative pathway

The aim of this study is to determine whether myeloma and lymphoma tumor cells can function as efficient antigen presenting cells (APC) to enhance the co-stimulation of T cells. The expression and function of T cell activation-related molecules, especially CD80, CD28, CD40 and CD40 ligand (CD40L), were studied on nine human myeloma cell lines (HMCL) and two B lymphoma cell lines. In the case of myeloma cell lines, the cells generally lacked CD80 antigen and expressed a heterogeneous CD40, and the expressions of CD40 and CD80 molecules could not be induced by either CD28 stimulation or CD40 ligation. Conversely, in the two B lymphoma cell lines, tumor cells expressed both CD80 and CD40 to some extent. CD28 stimulation could obviously increase the expression of CD80, CD40 and some adhesion molecules, and therefore generate a more efficient anti- tumor cell immunity. In conclusion, CD28 stimulation combined with CD40 antibody or soluble CD40 ligand may be a promising immunotherapeutic approach to B lymphoma.     

抗CD71人-鼠嵌合抗体的制备及其诱导肿瘤细胞凋亡

目的 :研究在液氮中冻存 2年的转染瘤细胞株 (D2C)复苏后其分泌抗CD71人 -鼠嵌合抗体的特异性、分泌量及诱导肿瘤细胞凋亡的效应。方法 :用ELISA方法检测转染瘤细胞培养上清的抗体分泌量。经DEAE SephadexA 5 0离子交换法纯化产生的抗体 ,并采用SDS PAGE电泳鉴定 ;台盼蓝拒染法观察其对K5 6 2细胞生存的影响 ;细胞计数和MTT试验测定嵌合抗体及鼠源性抗体对K5 6 2细胞的作用 ;琼脂糖凝胶电泳和透射电镜观察K5 6 2细胞凋亡。结果 :Balb/c裸鼠腹腔接种转染瘤细胞后 ,每只裸鼠腹水量在 3～ 5mL。抗体经纯化 ,产量约为 1～ 2mg/mL腹水。经SDS PAGE电泳鉴定 ,在分子量 5 5kD和 2 7kD处 ,可见有抗体IgG重链和轻链的染色条带。抗CD71嵌合抗体及鼠源性抗CD71抗体均可抑制K5 6 2细胞的增殖作用 ,且二者抑制作用无显著性差异 (P >0 .0 5 )。经琼脂糖凝胶电泳和透射电镜观察分别可见抗体诱导的K5 6 2细胞DNA梯形条带和细胞凋亡的形态改变。结论 :液氮中冻存 2年的转染瘤细胞体外生长良好 ,抗体分泌稳定 ,特异性高 ,并可诱导K5 6 2人红白血病细胞凋亡     

史氏鲟（Acipenser schrenckii）两种促性腺激素β亚基的原核表达

 采用遗传工程方法,重组表达史氏鲟两种促性腺激素β亚基蛋白。选用原核表达载体pET－22b(+),分别插入史氏鲟GtHⅠ&;Ⅱβ亚基成熟肽cDNA序列,构建成C端含有6个组氨酸(6-His)融合蛋白标签的表达质粒;分别转化大肠杆菌表达菌株BL21（DE3）并诱导表达2个基因。SDS-聚丙烯酰胺凝胶电泳显示重组融合蛋白相对分子质量分别为：GtHⅠβ亚基大约14 000,GtHⅡβ亚基15 000左右。分别用抗6个组氨酸融合标签的单克隆抗体及兔抗鲟鱼GTH多克隆抗体对2个表达蛋白进行Western Blot分析,结果显示重组蛋白表达正确且有较高的免疫活性。GTHⅠβ重组蛋白在2 h就有明显的表达,6 h后随着时间增加表达量不再增加;25 ℃诱导重组蛋白产量比37 ℃诱导产量低。获得的重组蛋白质将可用于建立鲟鱼GtH的放射免疫测定方法。     

胃印戒细胞癌中CD44v6，Survivin与PTEN的表达及临床病理学意义

探讨黏附相关因子变异体CD44v6、凋亡抑制基因Survivin和抑癌基因FIEN在胃印戒细胞癌组织中的表达与其临床病理特征之间的关系,及三者的关系。表明应用免疫组织化学SP法,检测CD44v6、Survivin和FIEN蛋白在73例胃印戒细胞癌组织中的表达。结果胃印戒细胞癌中CD44v6、Survivin和PIEN蛋白的阳性表达率分别为78．1％（57／73）、63．0％（46／73）、34．2％（25／73）。CD44v6随胃印戒细胞癌浸润深度加深、淋巴结的转移逐渐上升（P〈0．05）;随浸润深度加深、淋巴结的转移、器官转移、临床分期增加Survivin蛋白的阳性表达率逐渐上升（P〈0．01）,而FTEN基因的阳性表达率逐渐降低（P〈0．05）。在胃印戒细胞癌组织中CD44v6与PFEN蛋白的表达呈负相关（相关系数r=-0．214,P〈0．05）,Survivin与PTEN的表达呈负相关（r=-0．404,P〈0．01）,CD44v6与Survivin的表达呈正相关（r=0．28,P〈0．01）。因此可知,CD44v6、Survivin和PIEN基因的异常表达与胃印戒细胞癌临床病理特征有关,PIEN和Survivin基因表达可以作为判断胃印戒细胞癌生物学行为的指标;CD44v6与Survivin的协同作用,以及两者与PTEN拮抗作用共同参与胃印细胞癌的发生发展过程。     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.     

The structure of complement C3b provides insights into complement activation and regulation

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.     

Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules.

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).     

暗纹东方纯CD8α基因克隆、原核表达与多克隆抗体制备

CD8是与Ⅰ型主要组织相容性复合体（MHCI）结合,是T细胞表面受体（TCR）的共受体．也是T淋巴细胞表面的重要标志物。该研究报道了暗纹东方纯胸腺等组织中克隆获得CD8αcDNA序列及其相应的基因组序列。克隆到的cDNA序列全长1061bp,包含1个657bp的开放读码框（ORF）,编码218个氨基酸;其对应的基因组序列为1533bp,包含5个内含子和6个外显子。生物信息学分析表明,D8d蛋白质序列由信号肽、胞外可变区、铰链区、跨膜区和胞内区5部分组成。胞外区的可变区和铰链区部分各有两个高度保守的半胱氨酸残基,可能参与链内和链间二硫键的形成。氨基酸序列多重比对表明,暗纹东方鲍与其他鱼类的CD80α,特别是与鲽形目鱼类具有较高的同源性。为进一步研究暗纹东方纯CD8的生物学功能,构建了表达CD8α成熟肽胞外区的重组质粒,诱导表达出重组蛋白。以纯化的重组蛋白为抗原免疫大白兔,制备了抗血清。经间接ELISA法检测抗体效价表明。获得了高效价的特异性暗纹东方纯CD8c~抗体,为进一步研究CD8在鱼类淋巴细胞进化和适应性免疫中的作用奠定了基础。     

Efficacy of anti-CD20 chimeric Fab’ fragment on proliferation of B lymphoma cells

The variable domain of heavy chain (V_H) and light chain (V_L) genes of anti-CD20 monoclonal antibody HI⁴⁷ were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI⁴⁷ to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC₅₀ = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, K_a was about 8.9×10⁸ (mol/L)⁻¹.