金鱼H1组蛋白基因及其N端片段的克隆、表达及抗菌活性

用RT—PCR方法从金鱼体内成功分离得到编码H1组蛋白的基因，基因登录号AY184811。该基因与来源于大西洋鲑鱼的抗菌蛋白-H1组蛋白有极高的同源性（78％）。将全长基因及其N端（2—38氨基酸）序列成功克隆于毕赤酵母分泌表达载体pPIC9K并表达；表达产物初步检测有抗菌活性。结果表明，金鱼H1组蛋白为一种新发现的抗菌蛋白，其N端来源肽（AEVAPAASAPPAKAPKKKSAAKA KKAGPAVGDLIVKA）为抗微生物肽。金鱼H1组蛋白及其来源肽在金鱼先天免疫防御中发挥作用。     

Ⅰ型单纯疱疹病毒糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

目的构建单纯疱疹病毒Ⅰ型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础.方法PCR扩增HSV1-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K-gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性.结果获得了重组的酵母表达载体pPIC9K-gD.测序结果证实为HSV1-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量为40.52 ku,pI为7.67,包含完整成熟肽分值达1.7的多个抗原决定簇.结论成功构建了HSV1-gD成熟肽基因的毕赤酵母表达载体.     

复合干扰素在毕赤酵母中的分泌表达、纯化及活性分析

目的 在毕赤酵母中高效分泌表达复合干扰素。方法 根据毕赤酵母密码子偏性合成了复合干扰素基因，克隆至分泌型酵母表达载体pMKX9K，线性化后转化毕赤酵母GS115，筛选高表达菌株。诱导后的培养上清经过离子交换，疏水层析，凝胶过滤三步层析纯化，用细胞病变抑制法测定其活性，并结合Iowry法蛋白定量计算其比活性。结果 SDS-PAGE分析显示，重组蛋白以可溶性分子的形式存在于上清液中，经纯化后纯度达到96％，其N端氨基酸序列与理论值一致，比活与干复津相当。结论 复合干扰素在毕赤酵母中获得高效分泌表达，为获得大量基因工程产品奠定了基础。     

人类肝素酶B细胞表位的预测和免疫原性鉴定

目的 预测并鉴定肝素酶(heparanase)蛋白B细胞表位免疫原性.方法 以肝素酶蛋白的氨基酸序列为基础,采用DNAStar分析软件以及Bcepred在线二级结构分析工具分析其蛋白二级结构并预测B细胞表位.根据预测结果 ,采用8分支多抗原肽结构合成针对该表位的抗原肽,将后者与通用型T辅助表位人IL-1β线性短肽(VQGEESNDK,氨基酸163～171)联合免疫日本白毛黑眼兔,检测免疫血清效价,鉴定其特异性和免疫原性.结果 软件预测显示,肝素酶蛋白大亚基的第1～15位(MAP1)、第279～293位(MAP2)及175～189位(MAP3)氨基酸序列最可能为其优势B细胞表位.间接酶联免疫吸附试验、免疫印迹及免疫组化分析,证实MAP1、MAP2及MAP3均能诱导机体产生高滴度抗体,但仅MAP1、MAP2抗体具有高特异性,MAP2抗体与肝癌组织的结合力最强.结论 肝素酶大亚基的第1～15位、第279～293位氨基酸为其优势B细胞表位,其中第279～293位氨基酸的免疫原性最强,这为肝素酶多肽抗体及B细胞优势短肽疫苗研制提供了理论依据.     

人ZP3 186S突变蛋白在毕赤酵母X33细胞中的表达

目的:利用毕赤酵母表达人ZP3186S蛋白(23～348氨基酸),为临床相关疾病的诊断提供实验材料。方法:经PCR获得编码人ZP3(23～348氨基酸)的基因,构建表达载体pPICZα-hZP3。将ZP3186位点精氨酸突变为丝氨酸构建pPICZα-hZP3186S质粒。转染毕赤酵母X33细胞,用高浓度Zeocin筛选转染细胞,甲醇诱导目的蛋白分泌,通过SDS-PAGE和Western blot鉴定人ZP3186S蛋白。结果:pPICZα-hZP3186S表达质粒经测序正确,并在毕赤酵母X33中能够高效表达。结论:毕赤酵母能够分泌性表达重组人ZP3186S蛋白(23～348氨基酸),进一步研究其生物学活性为研制不孕症诊断试剂和免疫避孕疫苗提供实验材料。     

肽抗生素hPAB-β在毕赤酵母中的表达与活性鉴定

目的 构建表达人源肽抗生素hPAB-β的重组毕赤酵母,为大量制备hPAB-β奠定基础.方法 用PCR及引物延伸法得到天然及优化的hPAB-β序列,克隆入pPIC9K质粒,转化DH5α大肠埃希菌,用酶切和核苷酸测序鉴定.重组质粒经SalⅠ酶线性化,整合入毕赤酵母GS115中,在DNA,mRNA及蛋白水平鉴定阳性重组子.结果 构建的含天然及优化序列的重组质粒酶切均可得到相应大小的插入片段,与预期相符,测序结果表明序列和阅读框均正确.线性化的重组质粒转入GS115后,得到7个不同的高拷贝阳性转化子,均能表达重组肽并具有良好的抑菌活性.结论 本研究成功构建含天然及优化hPAB-β序列的重组毕赤酵母工程菌,能够用于肽抗生素hPAB-β的制备.     

HMGN2抗菌结构域的研究

目的：本室从人外周血单个核白细胞分离获得一个新的抗菌多肽。其氮端氨基酸序列与人HMGN2相同。本研究HMGN2的抗菌谱和其功能结构域。方法：OMIGA蛋白结构软件分析HMGN2的二维结构。合成HMGN2氮端和碳端肽片段和α-螺旋结构域。检测各合成肽片段的抗菌活性。同时构建HMGN2全肽和α-螺旋结构域编码基因原核表达载体及制备其大肠杆菌重组表达产品。检测重组全肽和α-螺旋结构域的抗菌活性。结果：0MIGA蛋白结构软件分析发现HMGN2含1个从其氨基酸序列第17位到47位的跨膜α-螺旋结构域。     

诱导毕赤酵母分泌表达致倦库蚊防御素Defensin成熟肽

目的诱导毕赤酵母(Pichia pastoris)分泌表达表达致倦库蚊(Culex quinquefasciatus)防御素(defensin)成熟肽,为进一步研究致倦库蚊defensin的抗病原作用、机理及其应用奠定基础。方法利用生物信息和RT-PCR技术克隆了编码致倦库蚊防御素的成熟肽全基因序列,并克隆到毕赤酵母甲醇诱导型分泌表达载体pPICZα-A的α-factor信号肽序列的下游,构建成pPICZα-A-defensin重组表达载体,表达载体经SacⅠ线性化处理后电击转化毕赤酵母X-33感受态细胞,转化子经Zeocin抗性筛选,菌落PCR,Mut表型鉴定和Tricine-SDS-PAGE分析,成功获得了能够有效表达重组defensin的pPICZα-A-Defensin/X-33甲醇利用表型工程菌。结果在甲醇诱导24 h后pPICZα-A-Defensin/X-33的工程菌分泌表达了预期Mr8 200大小的致倦库蚊防御素成熟重组肽。结论致倦库蚊防御素可以在毕赤酵母中诱导分泌表达。     

小鼠抗人C5aR短肽（9—30）单克隆抗体制备及鉴定

目的获得有生物功能的小鼠抗人 C5 a R短肽单克隆抗体。方法对 C5 a受体 (CD88)二级结构和 B细胞表位进行分析研究 ,采用 Fmoc方案固相合成 C5 a R N-端第 9～ 30位氨基酸残基的 2 2 -肽 ,以此为抗原免疫 Balb/ c小鼠。结果建立了 1株小鼠抗人 C5 a R短肽杂交瘤细胞系 E3,其平均染色体数目为 10 2条 ,所分泌抗体为 Ig G1,κ型 ,腹水抗体效价为 1×10 - 4 ～ 1× 10 - 6 ,可识别 U 937、人脐静脉内皮细胞 (VEC)和 PMN等表达 C5 a R细胞。 E3单抗的亲和常数 Ka=2 .5× 10 5 ,其结合表位为 C5 a R第 15～ 2 1位氨基酸基序 D1 5 DKDTL2 0 D。结论 B细胞表位多肽具有免疫原性 ,可制作单克隆抗体 ,为 C5 a-C5 a R相互作用的研究以及 AL I、ARDS等 C5 a相关疾病的研究提供实验材料。     

重组表达人载脂蛋白(a)羧基末端kringle结构域抑制新生血管

 目的 利用毕赤酵母重组表达人载脂蛋白（a）[Apo(a)]羧基末端kringle结构域,明确其抑制新生血管和肿瘤细胞增殖的能力。方法 分别构建重组表达Apo(a)羧基末端kringle V结构域（RHAKA）与kringle IV 10型 - kringle V结构域（RHAKB）的pPICZ?A质粒;转染毕赤酵母X-33分泌表达RHAKA与RHAKB,RHAKs利用His?Tag亲和层析纯化,以及反相高效液相色谱与氨基酸残基测序鉴定;明确RHAKs的糖基化及二硫键形成情况后,利用细胞增殖实验与鸡胚绒毛尿囊膜（CAM）实验检测RHAKs对新生血管和肿瘤细胞增殖的影响。结果 毕赤酵母可以大量表达RHAKs,并对RHAKs进行翻译后修饰;RHAKA与RHAKB可以抑制血管内皮细胞的增殖和CAM的新生血管,但对肿瘤细胞增殖无直接的抑制作用。结论 利用毕赤酵母高效重组表达人Apo(a)羧基末端kringle结构域可以显著抑制新生血管。     

重组广州管圆线虫半胱氨酸蛋白酶抑制剂（AcCystatin）的理化性质研究

目的研究重组广州管圆线虫半胱氨酸蛋白酶抑制剂（AcCystatin）的理化性质及其生物学功能。方法原核表达质粒pGEX-4T-1-AcCystatin经诱导表达、纯化酶切后获得纯化AcCystatin,采用Edman降解法N-端氨基酸测序进行重组蛋白鉴定。分别检测其紫外光谱、荧光光谱、圆二色谱和对人源Cathepsin B、Cathepsin G、Cathepsin L和Cathepsin S酶的抑制活性,分析AcCystatin理化性质。结果重组AcCystatin蛋白经纯化、酶切后相对分子质量约为13600,纯化效果良好,氨基酸测序结果与理论氨基酸序列完全相同。光谱学检测结果显示重组AcCystatin可形成二硫键,二级结构中α-螺旋、β-折叠和无规卷曲分别占39.57%、35.28%和25.25%。酶抑制实验结果显示AcCystatin可显著抑制Cathepsin B、Cathepsin L和Cathepsin S酶的活性,但对CathepsinG酶活性无明显作用。结论获得的可溶性AcCystatin重组蛋白产量大、纯度高、蛋白结构折叠正常,具有良好的生物学活性,为AcCystatin免疫调节机制的深入研究奠定了实验基础。     

Chemotactic response of human monocytes to pentapeptide analog derived from immunodeficiency virus protein gp 120

The octapeptide sequence of peptide T is contained within the envelope of HIV and seems to mediate the viral binding to CD4 expressing cells, including monocytes. The biological activity of the -aminobutyric acid pentapeptide derived from the C-terminal sequence of peptide T, in which the polar side chain of threonine in position 4 is substituted by a hydrophilic group, is measured by the monocyte chemotaxis assay. The ehemotactic activity of human monocytes is assessed by determining the concentration at which the pentapeptide analog is maximally active and the effectiveness at that concentration, in comparison with peptide T and two shorter homologs, the pentapeptide and tetrapeptide. These experiments suggest that the synthetic analog is a potent ehemotactic factor active at picomolar concentrations and that it competes with peptide T for the monocyte binding site.     

一种新型重组人源性白细胞介素-8活性检测方法的建立

目的通过蛋白质片段互补检测（proteinfragmentcomplementationassay,PCA）技术检测重组人源性IL-8的生物学活性。方法利用PCA方法构建了Split—TEVGPCR激活检测系统,为了验证其有效性,在不同的宿主系统中表达和纯化了各种亚型的人源性IL-8重组蛋白用于进行活性检测。蛋白样品包括：①IL-8亚型I（残基21-99）,通过在哺乳动物细胞HEK293中分泌表达前体IL-8基因获得,其N端信号肽（残基1—20）被切除;②IL-8亚型Ⅱ（残基23—99）和亚型Ⅲ（残基28—99）,通过在大肠杆菌BL21（DE3）中表达获得。结果Split—TEVGPCR激活检测系统证明纯化后的重组人源性IL．8样品对天然受体IL8RB都具有明显的激活活性,其EC50值分别为：12．32±0．89ng／mL（亚型I）、15．14±1．84ng／mL（亚型Ⅱ）和2．854-0．50ng／mL（亚型Ⅲ）。结论成功建立了-种新型的重组人IL-8活性检测方法,为进一步的理论研究和IL-8中和抗体的高通量筛选奠定了基础。     

N-亚甲基磷酸化壳聚糖与DNA相互作用的研究

目的探讨双亲性壳聚糖衍生物与质粒DNA通过自组装的方式形成基因纳米粒子的可行性,考察载体与质粒DNA的相互作用机制,为开发安全高效的非病毒载体提供新的途径。方法合成了一种双亲性壳聚糖衍生物N-亚甲基磷酸化壳聚糖(NMPCS),利用复凝聚的方法制备NMPCS/DNA纳米粒子复合物。用傅立叶变换红外光谱、核磁共振谱等手段对NMPCS进行了表征,通过凝胶电泳阻滞实验、动态光散射、原子力显微镜、圆二色谱等实验对NMPCS与质粒DNA之间的相互作用进行研究。结果经傅立叶变换红外光谱及核磁共振谱分析表明,改性后的壳聚糖的特征基团有显著变化,证明了亚甲基磷酸基团成功的引入。NMPCS与DNA能自组装形成类似球形的基因纳米粒子。结论壳聚糖基双亲性分子可能与细胞膜中的双亲性分子具有潜在相容性,使得载体/DNA复合物通过一种膜的去组装过程跨越细胞膜而最终表达,从而有望研制出高效的非病毒载体。     

Selection of phage-display peptides that bind specifically to the outer coat protein of Rice black streaked dwarf virus

Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.     

吉林省2013年麻疹病毒核蛋白基因推导的氨基酸位点变异分析

目的 研究吉林省2013年麻疹病毒核蛋白（Nucleoprotein,NP）氨基酸位点的变异.方法 对吉林省2013年麻疹病例咽拭子标本用逆转录-聚合酶链反应（RT-PCR）扩增NP基因片段,将麻疹病毒RT-PCR阳性产物进行测序,分析其NP基因推导的氨基酸序列变异情况.结果 吉林省2013年麻疹病毒均为H1a基因亚型.以吉林省2001年麻疹病毒NP基因推导的氨基酸序列为基准,2013年50份麻疹病毒RT-PCR阳性标本中有部分标本在4个氨基酸位点出现变异;在其中3个出现变异的氨基酸位点422位点（G→S）、457位点（S→G）、497位点（R→K）上,2001-2008年33株麻疹病毒中仅有个别毒株出现该3个位点的变异,而2009年、2010年、2013年在该3个位点发生如上变异的麻疹毒株所占比例分别为100％、76.9％以及32.0％ ～32.6％;在第430位点,仅2013年有44.0％的麻疹病毒阳性标本出现P→S的变异.结论 吉林省2013年麻疹病毒呈现NP的氨基酸位点变异多样化,提示要关注NP的氨基酸变异趋势.     

美托洛尔对CHF患者血浆BNP和TNF-α的影响

目的:探讨慢性心力衰竭(CHF)患者口服美托洛尔后血浆脑钠肽(BNP)和肿瘤坏死因子-α(TNF-α)变化的临床意义。方法:观察32例CHF患者(以下简称CHF组)给予美托洛尔治疗12周前后血浆BNP和TNF-α浓度的变化,并与30例体检健康者(以下简称对照组)作比较。血浆BNP、TNF-α采用生物活性法测定。结果:CHF组基础血浆BNP和TNF-α浓度非常显著地高于对照组(P<0.01),服药12周后,CHF组血浆BNP和TNF-d浓度显著降低(P<0.05)。结论:CHF患者服用美托洛尔后血浆BNP和TNF-α浓度降低。     

Use of flanking sequences to study secondary structure-activity correlations of a Mycobacterium leprae T cell epitope.

The 65-kDa protein of the intracellular pathogen M. leprae is prominent in the immune response to this mycobacterium, and individual T cell epitopes from this protein sequence have been defined. We have tested the stimulatory activity of extended analogs of the minimal peptide representing one such epitope, LQAAPALDKL, with a variety of tetrapeptide extensions added to enhance or destabilize alpha helix formation. The conformational potential of the peptides was measured by circular dichroism using aqueous trifluoroethanol as a secondary structure inducer. Although analogs with high helical potential activated T cells at low concentrations, a less helical variant was similarly potent. Activity also did not correlate with predicted overall alpha helical amphipathicity. One analog was found which stimulated T cell proliferation in the 50 pM range. The effect of tetrapeptide extensions on epitope activity is not consistent with the importance in activity of only a single stable secondary structure such as an alpha helix.     

Evaluation of the C-terminal C5a effector site with short synthetic C5a analog peptides

Biological activities have been determined for a series of 18 peptides based on the C-terminal sequence of human or rat C5a. Lysosomal enzyme release was tested in two cell types, the promyelotic leukemia cell line U937 and polymorphonuclear leukocytes. In addition, an ATP-release assay with guinea pig platelets was performed. It was demonstrated that the C-terminal octapeptide 67-74 of human C5a represents the minimal sequence required to induce a measurable biological signal in all assays. Extending this peptide to a length of 21 amino acids produced at best only a slight enhancement of potency. Amino acid replacements with either tryptophanyl or phenylalanyl residues in positions between 65–69 either increased potency (at position 67), or abrogated potency (at position 66) in the two lysosomal enzyme assays. N-terminal acylation with the fluorenylmethoxy-carbonyl-aminohexanoyl group slightly enhanced C5a potency. In desensitization experiments with guinea pig platelets all peptides with a C5a activity were able to desensitize not only the C5a but also the C3a responses.     

Structural and biological properties of Cucumber mosaic virus particles carrying hepatitis C virus-derived epitopes

The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.