精制狂犬病疫苗纯化方法的比较研究

地鼠肾细胞狂犬病疫苗原液经１００ ｋＤ 膜浓缩 ３０ 倍,分别选用（１）ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析法;（２）Ｓｅｐｈａｃｒｙ１ Ｓ－２００ ＨＲ 分子筛选层析法;（３）二次蔗糖等密度区带离心法对其进行纯化。用此三种方法各试制３ 批精制疫苗,结果表明,经ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析纯化后疫苗总蛋白含量减少９９％ 以上,抗原比活性提高１５９ 倍,抗原回收率达５０％ ,纯化疫苗以ＮＩＨ 法效力测定平均为５．４ ＩＵ／２ｍ ｌ;经Ｓｅｐｈａｃｒｙ１ Ｓ－２００ＨＲ 分子筛层析纯化后疫苗总蛋白含量减少 ９８％ 以上,抗原比活性提高４１ 倍,抗原回收率达６３％ ,纯化疫苗效力平均为６．２５ ＩＵ／２ｍ ｌ;经一次蔗糖等密度区带离心法纯化后疫苗总蛋白含量减少９８％ 以上,抗原比活性提高３２１ 倍,抗原回收率达４３％ ,纯化疫苗效力平均为６．１８ ＩＵ／２ｍ ｌ,三种纯化疫苗均符合Ｗ ＨＯ 规程要求。     

神经营养因子GDNF编码顺序的克隆及表达产物的纯化

根据基因库中的顺序,设计了胶质细胞源神经营养因子（ＧＤＮＦ）基因的ＰＣＲ引物,以此从人基因组ＤＮＡ中扩增并克隆了ＧＤＮＦ的编码序列,经ＤＮＡ测序确认后,该片段克隆到表达质粒ｐＥＴ－３ａ中,转化大肠杆菌ＢＬ２１（ＤＥ３）．培养的重组菌经ＩＰＴＧ诱导,在Ｔ７启动子调控下表达出ｈＧＤＮＦ蛋白．经电泳分析表明ＧＤＮＦ主要存在于细菌包涵体中．从培养菌中制备包涵体,经充分洗涤,溶解于含８ｍｏｌ／Ｌ尿素的变性缓冲液中．经ＳＰ－Ｓｅｐｈａｒｏｓｅ柱层析分离,梯度洗脱,以１５％ＳＤＳ－ＰＡＧＥ检查含ＧＤＮＦ的部分．将含单体ＧＤＮＦ部分进行复性,再次用ＳＰ－Ｓｅｐｈａｒｏｓｅ离子柱分离同源二体ＧＤＮＦ．最后经ＳＤＳ－ＰＡＧＥ制备电泳纯化,纯度大于９５％．经Ｎ端测序表明序列正确．经测定,每升培养菌可得约１０ｍｇ纯化的ＧＤＮＦ．     

应用SDS-PAGE及光密度扫描测定肾综合征出血热纯化疫苗(Ⅰ型)纯度

应用ＳＤＳ－ＰＡＧＥ后光密度扫描的方法,对兰州生物制品研究所生产的５批肾综合征出血热（ＨＦＲＳ）乳鼠脑Ⅰ型纯化疫苗进行了纯度测定,结果５批疫苗的纯度为７５．７％－８９．８％,平均为８１．６８％,说明兰州所生产的乳鼠脑纯化疫苗纯度较好,杂蛋白含量很少,为精制纯化疫苗     

应用SDS—PAGE及光密度扫描测定肾综合征出血热纯化疫苗（？…

应用ＳＤＳ－ＰＡＧＥ后光密度扫描的方法,对兰州生物制品研究所生产的５批肾综合征出血热（ＨＦＲＳ）乳鼠脑Ⅰ型纯化疫苗进行了纯度测定,结果５批疫苗的纯度为７５．７％￣８９．８％,平均为８１．６８％,说明兰州所生产的乳鼠脑纯化疫苗纯度较好,杂蛋白含量很少。为精制纯化疫苗。     

人GM—CSF cDNA的克隆和在大肠杆菌中的表达

从诱导的人胚肺细胞ＨＦＬ株中提取总ＲＮＡ．经ＲＴ－ＰＣＲ反应获取了人ＧＭ－ＣＳＦｃＤＮＡ,ＤＮＡ序列测定表明其顺序与文献报道完全一致。为了获得高效表达,应用ＰＣＲ改造了人ＧＭ－ＣＳＦ的ｃＤＮＡ５’端核苷酸序列,并将改造的人ＧＭ－ＣＳＦ基因插入含Ｔ７启动子的质粒ｐＥＴ－１１ｄ构建成表达质粒ｐＥＴＣ－５,将此质粒转化大肠杆菌株ＢＬ２１（ＤＥ３）得到表达菌株ＢＬＥＣ４。表达菌株用０．５ｍｏｌ／ＬＩＰＴＧ诱导２小时后,产生大量重组蛋白并形成包涵体。ＳＤＳ—ＰＡＧＥ电泳图谱扫描结果表明,ｒｈＧＭ－ＣＳＦ产量占菌体总蛋白量的１６％。ＥＬＩＳＡ和ＴＦ－１细胞培养测定表明,初步纯化和复性的ｒｈＧＭ－ＣＳＦ具有天然的ｈＧＭ－ＣＳＦ生物活性。     

国产重组酵母乙型肝炎疫苗接种小学生的3年免疫效果观察

本文报告对２５８ 名小学生应用０、１、６ 月程序,接种２ 批国产酵母疫苗（５ μｇ／０．５ ｍ ｌ）,１ 批Ａｍ ｇｅｎ 酵母疫苗（１０ μｇ／ｍ ｌ）,１ 批Ｍ ＳＤ 酵母疫苗（５ μｇ／０．５ ｍ ｌ）的小学生免后３ 年（（Ｔ３６）效果观察。结果表明,Ｔ３６ 时抗体 ＧＭＴ（几何平均滴度）,Ａｍ ｇｅｎ 疫苗组（１４５．７５）显著高于 ２ 批国产疫苗（９２．１１、８３．５２）和Ｍ．Ｓ．Ｄ 苗组（７４．６２）,抗体ＧＭＴ 峰值显著低于Ａｍ ｇｅｎ 和Ｍ ．Ｓ．Ｄ 苗的２ 批国产酵母疫苗,与Ｍ．Ｓ．Ｄ 苗抗体ＧＭＴ 水平无显著差异（Ｐ＞ ０．０５）。抗体阳转率间各疫苗组均无显著差异（９３．１０％ ～７４．１４％ ）。本次随访结果表明,采用０,１,６ 免疫程序,国产酵母疫苗免后３ 年的抗体阳转率和抗体ＧＭＴ水平不低于进口同类酵母疫苗的水平。     

兔出血症病毒结构多肽分析

粗提病毒经Ｓｅｐｈａｒｏｓｅ４Ｂ层析后,获得纯化的兔出血症病毒（ＲＨＤＶ）。提纯病毒经过ＳＤＳ－ＰＡＧＥ经考马斯亮蓝染色显示Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ７条多肽,凝胶扫描显示Ａ为ＲＨＤＶ主要结构多肽。用多抗和单抗作免疫转印分析,证实Ａ、Ｂ、Ｃ、Ｄ、Ｅ、和Ｇ为结构多肽,此６条结构多肽间的抗原关系十分密切。     

Mn-SOD与抗癌胚抗原单链抗体基因的融合及高效表达

将Ｍｎ－ＳＯＤ与抗癌胚抗原（ＣＥＡ）单链抗体基因（Ｓｃ－Ｆｖ ｇｅｎｅ）融合,重组到含Ｔ７启动子的表达载体ｐＥＴ－２２ｂ（＋）中,构建表达质粒ｐＥＴＭｎ－ＳＯＤ－ＳｃＦｖ,并转化大肠杆菌ＢＬ２１（ＤＥ３）,进行高效表达,表达物占菌体可溶性总蛋白的２４％。ＳＤＳ－ＰＡＧＥ和蛋白质和迹图谱显示表达物分子量为４５ｋＤ与融合基因编码蛋白质的理论值相符。该蛋白质在大肠杆菌中为泌型表达有利于纯化。ＲＩＡ测定表     

重组人碱性成纤维细胞生长因子(bFGF)融合蛋白的高效表达及鉴定

碱性成纤维细胞生长因子（ｂＦＧＦ）参与了许多细胞生长和分化的调控过程。本文采用重组ＤＮＡ技术在大肠杆菌中高效表达了人ｂＦＧＦ。首先将编码人ｂＦＧＦ基因克隆到ｐＸＴ表达载体中与其上游的一短Ｓ导肽共一阅读框架,ｂＦＧＦ基因的表达受强的Ｔ７启动子调控。采用ＢＬ２１（ＤＥ３）大肠杆菌作为宿主菌,用ＩＰＴＧ诱导ＢＬ２１（ＤＥ３）细菌合成的Ｔ７ＲＮＡ聚合酶,后者可催化高水平的ｂＦＧＦ基因表达,其ｂＦＧＦ产量可占总菌体蛋白的４２.５％。采用肝素Ｓｅｐｈａｒｏｓｅ一步亲和层析法直接从诱导后的细菌裂解产物中得到纯化的重组人ｂＦＧＦ蛋白。经Ｗｅｓｔｅｒｎ印迹分析证明该蛋白可被人ｂＦＧＦ特异性单克隆抗体所识别。进一步研究证明该蛋白具有刺激ＮＲ６Ｒ－３Ｔ３成纤维细胞增殖的生物学活性,并且这一活性可被人ｂＦＧＦ特异性中和抗体所中和。     

用酵母双杂交系统研究Smad3和Smad4的相互作用

Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 是将 Ｔ Ｇ Ｆ β的信号从细胞外传递到细胞核内的重要的信号传导蛋白． Ｔ Ｇ Ｆ β与其受体结合后,激活受体的磷酸激酶,使 Ｓｍ ａｄ３ 发生磷酸化,活化的 Ｓｍ ａｄ３ 与 Ｓｍ ａｄ４ 结合,形成异源复合物,进入到核中．然后 Ｓｍ ａｄ４ 以 Ｄ Ｎ Ａ 结合蛋白的形式与特定的 Ｄ Ｎ Ａ 结合,将 Ｔ Ｇ Ｆ β的信号传到核内．激活转录,诱导背中胚层的形成,抑制细胞的分化等．经研究利用酵母双杂交试验,鉴定了 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 相互作用的功能区域．构建 Ｓｍ ａｄ３ 和 Ｓｍ ａｄ４ 的 Ｃ 端、 Ｎ 端和中间连接区的突变体,将这些突变体克隆到 ｐ Ｇ Ａ Ｄ４２４ 和 ｐ Ｇ Ｂ Ｔ９ 载体中,并转化到 Ｈ Ｆ７ Ｃ 酵母中．通过 Ｌｅｕ－ ／ Ｔｒｐ－ ／ Ｈｉｓ－ Ｓ Ｄ 平板上菌落的形成,和 Ｘ－ ｇａｌ显色反应鉴定转化到酵母中的两个克隆质粒的相互作用．结果显示 Ｓｍ ａｄ４ 与 Ｓｍ ａｄ３ 异源相五作用时,主要是通过 Ｓｍ ａｄ４ 的中间连接区．在同源作用时, Ｓｍ ａｄ３ 是通过 Ｃ 端,而 Ｓｍ ａｄ４ 是通过中间连接区进行的．     

Assessment of Human Immune Response to Mutans Streptococcal Glucosyltransferase Peptides Selected by MHC Class II Binding Probability

Dental decay is a major public health challenge, causing substantial social and economic burdens. In animals, vaccination against mutans streptococci, the causative organism, interferes with dental caries. The mutans streptococcal glucosyltransferase (GTF) has been effectively used as a protein antigen in experimental dental caries vaccines. Compared to whole proteins, peptide subunits can focus immune responses on protective epitopes, and not on potentially harmful cross reactive antigens. In the past we selected peptide subunits of GTF for vaccine discovery based on putative functional significance and conservation of GTF primary structure. To focus on the immunogenicity of peptides, we estimated the probability of MHC class II binding. Twenty 20-mer linear GTF peptides were synthesized on this basis and their immunoreactivity explored. Significant human peripheral blood mononuclear cell (PBMC; n = 12) proliferation was observed in response to amino acids (AA) 502–521 (peptide 7), located in the catalytic domain of GTF. Human serum (n = 36) antibody reactivity was observed to AA 438–457 (peptide 5), AA 502–521 (peptide 7), and AA 1376–1395 (peptide 16). Whole saliva mutans streptococcal levels were used as markers of mutans infection, and dental examinations to determine existing and historic caries (DMFS score) were performed. DMFS scores correlated with mutans streptococcal counts, but not with immune responses. We have identified peptides with projected avid MHC-binding activity that reacted with human PBMC and serum antibody, implying that these peptides are immunogenic and may be of significance in a subunit dental caries vaccine.     

The scientific and public-health imperative for a vaccine against dental caries

Dental caries is caused by one of the most ubiquitous bacterial infections of humans. In many countries such as Brazil and China, this disease is reaching epidemic proportions, and it is clear that a more effective public-health measure to combat dental caries is needed, because disadvantaged children are the most severely affected. One of the main groups of oral microorganisms, the mutans streptococci, has been associated with the aetiology of dental caries, and preclinical studies of immunological interventions have shown the feasibility of interfering with this disease. Moreover, clinical trials have indicated that a mucosal immune response to a crucial antigen(s) of mutans streptococci can influence the pathogenesis of dental caries. Evidence that this antigen(s) is appropriate for use in a vaccine against dental caries, as well as evidence for an appropriate target population of individuals and a logical time of administration, has now emerged.     

Comparative study of the effect of green and roasted water extracts of mate (Ilex paraguariensis) on glucosyltransferase activity of Streptococcus mutans

Glucosyltransferase (GTF) plays an important role in the development of dental caries. This study was carried out to compare the efficiency of green mate (GM) and roasted mate (RM) water extracts, drinks rich in polyphenolic compounds consumed in the subtropical region of South America, on the extracellular GTF activity from Streptococcus mutans. The RM extract exhibited a greater inhibitory effect (IC?? of 10 mg/mL) despite presenting lower polyphenolic content. The kinetic analysis showed that there were significant differences (P?相似文献   

Exploring the sequence-structure protein landscape in the glycosyltransferase family

To understand the molecular basis of glycosyltransferases' (GTFs) catalytic mechanism, extensive structural information is required. Here, fold recognition methods were employed to assign 3D protein shapes (folds) to the currently known GTF sequences, available in public databases such as GenBank and Swissprot. First, GTF sequences were retrieved and classified into clusters, based on sequence similarity only. Intracluster sequence similarity was chosen sufficiently high to ensure that the same fold is found within a given cluster. Then, a representative sequence from each cluster was selected to compose a subset of GTF sequences. The members of this reduced set were processed by three different fold recognition methods: 3D-PSSM, FUGUE, and GeneFold. Finally, the results from different fold recognition methods were analyzed and compared to sequence-similarity search methods (i.e., BLAST and PSI-BLAST). It was established that the folds of about 70% of all currently known GTF sequences can be confidently assigned by fold recognition methods, a value which is higher than the fold identification rate based on sequence comparison alone (48% for BLAST and 64% for PSI-BLAST). The identified folds were submitted to 3D clustering, and we found that most of the GTF sequences adopt the typical GTF A or GTF B folds. Our results indicate a lack of evidence that new GTF folds (i.e., folds other than GTF A and B) exist. Based on cases where fold identification was not possible, we suggest several sequences as the most promising targets for a structural genomics initiative focused on the GTF protein family.     

Comparative study of the effect of green and roasted water extracts of mate (Ilex paraguariensis) on glucosyltransferase activity of Streptococcus mutans

Glucosyltransferase (GTF) plays an important role in the development of dental caries. This study was carried out to compare the efficiency of green mate (GM) and roasted mate (RM) water extracts, drinks rich in polyphenolic compounds consumed in the subtropical region of South America, on the extracellular GTF activity from Streptococcus mutans. The RM extract exhibited a greater inhibitory effect (IC₅₀ of 10?mg/mL) despite presenting lower polyphenolic content. The kinetic analysis showed that there were significant differences (P?<?0.05) between the extracts with respect to the values for K_m and K_i, whereas the values for V_max were the same, implying the competitive nature of GTF inhibition. GTF activity was also measured using selected polyphenols as inhibitors, and the most effective inhibitors were rutin and caffeoylshikimic acid. The characterization of the extracts by ESI-MS and UPLC-MS showed that the compounds formed during roasting, possibly shikimic acid derivatives and other unindentified compounds formed by the Maillard reaction, appeared to contribute to the inhibition of GTF activity.     

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.     

Quantifying beta-sheet stability by phage display

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.     

郑州孙庄遗址仰韶文化人群的龋病

孙庄遗址位于河南省郑州市中原区孙庄村,是分布在黄河中游的一处仰韶文化晚期遗址,该遗址出土的54例仰韶时期的人骨保存状况良好,为我们了解仰韶文化人群的龋患情况提供了珍贵的资料。本文以肉眼观察为主并结合超景深显微镜对遗址出土的846枚牙齿进行鉴定、统计与分析,得出以下结论：孙庄遗址古代人群的龋病患病率70.37%,龋齿率22.93%,龋均3.59;壮年组患龋率最高,为88.89%,不同年龄组之间的龋齿率差异性显著;龋齿率女性为30.55%,男性为16.16%,女性龋齿率显著高于男性,P(0.000)<0.05,两性之间的龋齿率有显著差异;浅龋率为6.70%,中龋率为21.65%,深龋率为27.32%,深龋已穿髓率为19.07%,残冠残根率为25.26%,随着年龄的增长,龋病的病变程度呈加重趋势;龋损范围小于1/2牙冠累龋最常见,占总患龋齿数的42.27%;上颌龋齿率为26.91%,下颌龋齿率为19.70%,上下颌龋齿率差异显著（P<0.05）。龋病在不同牙位上的发生率依次为M3>M2>P2>M1>P1>C>I1>I2;邻面和 面是主要的龋患分布处,分别占患龋牙齿数的46.40%和39.18%。孙庄人群门齿较高的患龋率表明该遗址人群患龋情况已经非常严重,患龋率与龋齿率明显高于其他古代组,较高的龋病罹患率可能与孙庄人群复杂的农业经济模式有关。     

A lemon myrtle extract inhibits glucosyltransferases activity of Streptococcus mutans

Streptococcus mutans is a bacterium found in human oral biofilms (dental plaques) that is associated with the development of dental caries. Glucosyltransferases (GTFs) are key enzymes involved in dental plaque formation, and compounds that inhibit their activities may prevent dental caries. We developed a screening system for GTF-inhibitory activities, and used it to profile 44 types of herbal tea extracts. Lemon myrtle (Backhousia citriodora) extract exhibited the highest GTF-inhibitory activity, with an IC₅₀ for GTF in solution of 0.14 mg mL^?1. Furthermore, lemon myrtle extracts had the third-highest polyphenol content of all tested extracts, and strongly inhibited S. mutans biofilm. Interestingly, lemon myrtle extracts did not inhibit cell growth.     

Differential immunogenicity of a DNA vaccine containing the Streptococcus mutans wall-associated protein A gene versus that containing a truncated derivative antigen A lacking in the hydrophobic carboxyterminal region

Two plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region.