变异链球菌表面蛋白PAcP与霍乱毒素B亚单位融合基因在转基因番茄中的表达

目的:在获得含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄原代种子的基础上,应用分子生物学技术检测外源基因在转基因植株中的表达,测定目的蛋白的含量。方法:PCR筛选含编码变异链球菌表面蛋白PAcP与霍乱毒素B亚单位融合基因的转基因番茄植株;提取番茄果实总蛋白,用BCA试剂盒测定番茄果实总蛋白含量;通过Western印迹检测外源蛋白的表达情况,并用ELISA法对外源目的蛋白含量进行测定。结果:PCR扩增分析可见1.6 kb特异性扩增条带,出现特异性条带的植株占总检测植株的55.6%;转基因番茄总蛋白含量为3.93 mg/mL,Western印迹结果显示,在PVDF膜上,约60 kD处出现特异性条带;ELISA测得表达的目的蛋白占番茄可溶性总蛋白的0.18%。结论:含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄子代植株能有效表达外源蛋白。     

转基因番茄防龋疫苗的基础研究Ⅰ.变形链球菌pac基因唾液粘附区植物表达质粒的构建

目的 :构建变形链球菌表面蛋白基因 (pac)唾液粘附区的植物表达质粒pROP1和pRPB1。方法 :用PCR扩增的含变形链球菌表面蛋白唾液粘附区P1片段 (184- 1946bp)与植物的高效表达质粒pROKCP结合 ,构建pROP1表达质粒 ;且将此质粒与Bar基因 (抗除草剂基因 )连接 ,构建表达质粒pRPB1,酶切电泳检测。结果 :从pPC41中扩增的P1片段整合到pROKCP的适当部位 ,构成重组质粒pROP1,从pPBar分离出的Bar基因整合到pROP1,构成重组质粒pRPB1。结论 :本实验成功地构建了携带变形链球菌表面蛋白唾液粘附区的植物表达质粒pROP1和pRPB1。     

转基因番茄防龋疫苗的基础研究：I.变形链球菌pac基因唾液粘附区 …

目的 构建变形链球菌表面蛋白基因（ｐａｃ）唾液粘附区的植物表达质粒ｐＲＯＰ１和ｐＲＰＢ１。方法 用ＰＣＲ扩增的含变形链球菌表面蛋白唾液粘附区Ｐ１片段（１８４－１９４６ｂｐ）与植物的高效表达质粒ｐＲＯＫＣＰ结合,构建ｐＲＯＰ１表达质粒;且将此质粒与Ｂａｒ基因（抗除草剂基因）连接,构建表达质粒ｐＲＰＢ１,酶切电泳检测。结果 从ｐＰＣ４１中扩增的Ｐ１片段整合到ｐＲＯＫＣＰ的适当部位,构成重组质粒ｐＲＯＰ     

变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因转基因黄瓜植株的获得及鉴定

目的:经农杆菌介导将变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因导入黄瓜,获得转基因黄瓜植株,为转基因可食防龋疫苗的研究提供实验基础。方法:利用双元载体pCAMBIA2301构建变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合质粒(PAcA-ctxB)的植物表达载体p2355-PAcA-CTB;电转化法导入农杆菌EHA105;采用叶盘转化法转化黄瓜,转基因植物经过卡那霉素抗性筛选、GUS基因染色、PCR及Southern blot杂交分析检测目的基因鉴定。结果:成功得到转基因黄瓜植株,卡那霉素抗性筛选、GUS基因染色、PCR及Southern blot杂交分析检测证实目的基因PAcA-ctxB已整合至黄瓜基因组中。结论:获得了含有变异链球菌表面蛋白A区与霍乱毒素B亚单位嵌合基因的转基因黄瓜植株,为转基因可食防龋疫苗的进一步研究提供了实验基础。     

变链菌葡糖基转移酶植物表达质粒p2355-gtfB、p2365-gtfB转化烟草研究

目的获得含变形链球菌葡糖基转移酶编码基因gtfB的转基因烟草植株。方法将携带变形链球菌葡糖基转移酶编码基因gtfB的植物表达载体p2355-gtfB、p2365-gtfB采用叶盘法经根癌农杆菌介导转化烟草,从而获得抗性烟草植株,并通过GUS染色、npt-Ⅱ基因和部分目的基因片段的PCR扩增对转基因烟草植株进行检测。结果抗性苗经GUS组织染色呈阳性,PCR扩增检测npt-Ⅱ基因及部分目的基因,表明获得了转基因烟草植株。结论目的基因已成功导入烟草基因组中,且具有快速鉴定转化株的优点。     

转基因番茄防龋疫苗中外源目的基因拷贝数的检测

目的：采用SYBR Green实时荧光定量 PCR方法检测转基因番茄防龋疫苗中外源目的基因的拷贝数。方法：用本课题组前期构建的重组质粒pEAC10、pEPC10作为标准品,SYBR Green实时荧光定量PCR法对含外源目的基因pacA-ctxB、pacP-ctxB的转基因番茄植株总基因组样本重复检测,取平均值作为目的基因拷贝数。结果：含pacA-ctxB转基因番茄植株外源目的基因的拷贝数为1.3,含pacP-ctxB转基因番茄植株外源目的基因的拷贝数为3.2。结论：构建的转基因番茄植株属低拷贝转基因植物,具有较好的稳定性。     

变形链球菌表面蛋白A区遗传多态性的研究

目的:探讨变形链球菌表面蛋白A区遗传多态性与其粘附性能的关系。方法:分别选取本实验室前期工作所获得的粘附能力较强和较弱的血清c型变形链球菌临床分离株，提取全菌DNA，经PCR扩增表面蛋白A区编码基因spaP-a后，用限制性内切酶Hae班进行限制性片段长度多态性分析。结果:经Hae ]II酶切后，两组血清c型变形链球菌均出现了4种基因型，且4种基因型在两组菌株的构成情况不同。结论:血清c型变形链球菌临床分离的 spaP-a具有遗传多态性，粘附能力较强的菌株比粘附能力较弱的菌株具有更明显的异质性。     

变形链球菌表面蛋白遗传多态性与龋病发生关系的研究Ⅱ表面蛋白V区、P区编码基因序列的测定

目的 :对不同粘附力变形链球菌临床分离株表面蛋白V区、P区编码基因进行序列测定。方法 :实验菌株选自本实验室前期工作所获得的spaP pv经AluI酶切后呈不同基因型的不同粘附力血清c型变形链球菌临床分离株。提取全菌DNA ,经PCR扩增表面蛋白V区、P区编码基因spaP pv( 2 0 60～ 3 15 7bp)后 ,进行序列测定。结果 :不同粘附力变链菌临床株spaP pv经AluI酶切后呈a、b 2种基因型的菌株测出的spaP pv序列均有 7个AluI酶切位点 5′ AG↓CT 3′。 10株a型菌株序列除了几个碱基点突变外均相同 ,9株b型菌株序列亦如此。a型有 2个DNA片段与b型不同 ,经分析 ,这 2个变异片段均位于V区。结论 :变链菌临床株表面蛋白粘附性能的差异可能是编码基因可变区域V区出现变异所致     

基因重组鼠伤寒沙门氏菌防龋疫苗的研究Ⅱ.携带变形链球菌pac基因表达质粒的构建与初步鉴定

目的：构建携带变形链球菌表面蛋白pac基因A区片段的原核高效表达系统。方法：对含变形链球菌表面蛋白pca基因的pPC41质粒进行PCR扩增,获得了pac基因A区片段,将扩增获得pac基因A区片段与表达质粒pET17-b定向克隆,重组质粒用B HI,EcoRV双酶切后电泳鉴定。结果：获得携带pac基因片段的重组持粒,结论：利用基因工程技术获得的重组质粒为后继工作准备了实验基础。     

重组质粒pCIA-P在大肠杆菌BL21(DE3)中的表达研究

目的　证实重组质粒 pCIA P可编码表达具有变形链球菌S .mutans表面蛋白PAc抗原性的表达产物。方法　在选择合适的原核表达系统后 ,诱导外源基因表达出目的蛋白 ,通过蛋白质电泳技术及免疫印迹法检测表达蛋白 ,以观察 pac基因重要抗原决定簇编码区DNA片段在原核细胞中的表达情况。结果　克隆片段在原核系统下可完成表达 ,表达产物保存了S .mutans表面抗原的免疫原性。结论　该实验为探索出一种简便快捷地检测与分析重组DNA疫苗表达产物的分子量及抗原性的方法提供了实验依据     

转基因番茄可食用防龋疫苗免疫BALB/c小鼠的实验研究

目的: 观察含PAcA/CTB的转基因番茄可食用防龋疫苗的免疫原性及免疫反应性。方法: 将18只6~8周龄的雄性BALB/c小鼠,随机分为3组,每周分别灌胃含有PAcA/CTB嵌合蛋白的转基因番茄果汁(实验组)、非转基因番茄果汁(阴性对照组)、灭活全菌疫苗(阳性对照组)。于首次免疫前和免疫后第1、2、3、4周采集血液、唾液样品。ELISA检测其血清和唾液样本中的抗变异链球菌PAcA的IgG、IgA抗体效价。结果: 阳性对照组和实验组小鼠血液和唾液中特异性抗体从免疫后1周开始升高,初始免疫后第4周达高峰,分别与阴性对照组比较存在统计学差异(P<0.05或P<0.01)。免疫后第2、3、4周阳性对照组与实验组差异有统计学意义(P<0.05)。结论: 转基因番茄所表达的外源目的蛋白具有抗原性,能够诱导BALB/c小鼠产生免疫应答。     

Deletion in sortase gene of Streptococcus mutans Ingbritt

Our previous studies on Streptococcus mutans have demonstrated that surface proteins containing a C-terminal sorting signal, such as surface protein antigen (PAc), glucan-binding protein C (GbpC) and dextranase (Dex), are anchored to the cell wall by a sortase (SrtA). In this study we found that, unlike other strains of S. mutans, strain Ingbritt did not exhibit cell wall-anchoring of PAc, GbpC and Dex. It is speculated that the SrtA of strain Ingbritt did not function in the cell wall-anchoring process of these surface proteins. Sequence analysis revealed a deletion of an 11-bp nucleotide sequence in the srtA gene of strain Ingbritt, resulting in the generation of a new termination codon, resulting in production of an incomplete SrtA enzyme protein. As a result, strain Ingbritt showed a localization change of PAc, GbpC and Dex in the cell, implying that strain Ingbritt loses the biological functions mediated by the cell surface-associated proteins of S. mutans. These results suggest that strain Ingbritt could be less cariogenic than other strains of S. mutans.     

A study on anti-caries activity of genetic recombinant vaccine of Streptococcus lactis. II. Immunization in vein with recombinant S. lactis in pregnant rabbits]

The effects of immunization in vein with recombinant S. lactis HL107 carring the S. mutans surface protein PAc gene in pregnant rabbits were studied. The results indicated that specific anti-PAc IgG in serum and milk were obviously induced 1 week after immunization and retained at high level for several weeks. It suggests that the recombinant S. lactis HL107 possessing immunogenicity of S. mutans surface protein PAc is able to stimulate specific systemic immune response against PAc.     

Contribution of cell surface protein antigen c of Streptococcus mutans to platelet aggregation

Introduction:  Streptococcus mutans is considered to be one of the pathogens that cause infective endocarditis. The purpose of the present study was to examine the properties of S. mutans with regard to platelet aggregation by focusing on its high molecular protein antigen c (PAc).
Methods:  The platelet aggregation properties of six clinical strains and one isogenic mutant strain of S. mutans were analysed using an aggregometer and confocal microscopy, as well as with an inhibition assay of platelet aggregation using anti-PAc serum.
Results:  S. mutans strains with PAc expression induced platelet aggregation, while a PAc-deficient mutant and two clinical isolates with no PAc expression did not. When platelets were pretreated with higher amounts of anti-PAc serum, the platelet aggregation rate was reduced in a dose-dependent manner, indicating that PAc binds directly to platelets.
Conclusion:  S. mutans PAc is involved in human platelet aggregation and may be one of the virulence factors in the pathogenesis of infective endocarditis.     

乳清抗体对变形链球菌粘附能力的影响

采用ＥＬＩＳＡ法检测重组乳链球菌ＨＬ１０７免疫后孕兔乳清特异性抗体水平，用＾３Ｈ－胸腺嘧啶同位素标记技术观察特异性抗体变形链球菌粘附能力的影响，结果表明，免疫后孕兔乳清中有高水平的特异性抗体产生，并且明显减少了变形链菌对唾液色被的羟基磷灰石的粘附，提示携带有变形链球菌表面蛋白抗原基因的重组乳链球菌ＨＬ１０７具有良好的免疫原性，乳清抗表面蛋白抗体降低了变形链球菌的粘附能力。     

转基因番茄可食用防龋疫苗免疫BALB/c鼠的实验初探

目的：观察转基因番茄可食用疫苗的免疫原性和免疫反应性。方法：将60只60d龄的雌性BALB／c小鼠随机分为4组，分别口服或腹膜下注射转基因番茄或对照番茄。35d后取小鼠血清样本，用ELISA的方法检测其血清样本中的抗变形链球菌PAc的IgG抗体效价。结果：口服和腹膜下注射转基因番茄组小鼠的血清中均出现了抗PAc的IgG抗体，分别与对照组相比具有统计学差异（P〈0．05），且口服转基因番茄组与注射转基因番茄组的小鼠血清抗体水平无统计学差异（P〉0．05）。结论：转基因番茄所表达的外源蛋白具有抗原性，能够诱导实验动物产生免疫应答。     

The sortase of Streptococcus mutans mediates cell wall anchoring of a surface protein antigen

Sortase has been shown to be a protease that catalyzes the cell wall anchoring of surface proteins containing an LPXTG motif in gram-positive bacteria. In this study, we determined the complete nucleotide sequence of the sortase gene (srtA) of Streptococcus mutans and found a surface protein that was linked to the cell wall by the sortase. The results show that srtA gene of S. mutans consisted of 741 bp and encoded for a sortase protein of 246 amino acids with a molecular weight of 27 489. The deduced amino acid sequence of the S. mutans sortase was highly homologous (65-58%) to those of other Streptococcal species. In a S. mutans mutant lacking sortase, two surface proteins of 200 and 75 kDa were released to the culture supernatant. Western blot analysis with specific antiserum showed that the 200 kDa protein was a surface protein antigen designated PAc. These results suggest that the sortase catalyzes anchoring of the antigen PAc to the cell wall.     

Inactivation of srtA gene of Streptococcus mutans inhibits dextran-dependent aggregation by glucan-binding protein C

A sortase-deficient mutant of Streptococcus mutans was prepared by insertional inactivation of a sortase gene (srtA). The srtA mutant was defective in cell wall-anchoring of two surface proteins 200 and 75 kDa in size. A previous study has shown that the 200 kDa protein is a surface protein antigen PAc and that the sortase catalyzes cell wall-anchoring of PAc in S. mutans. In this study another surface protein 75 kDa in size was examined by immunologic and physiologic methods. Western blot analysis with a specific antiserum showed that the 75 kDa protein was a surface protein, glucan-binding protein C. The protein was overexpressed under a stress condition including a sublethal concentration of tetracycline. The srtA mutant cells also lost the ability of dextran-dependent aggregation. These results suggest that the S. mutans sortase mediates cell wall-anchoring of the glucan-binding protein C and dextran-dependent aggregation of this organism.