血清TAP、proGRP、cyfra21-1与Ⅲ~Ⅳ期NSCLC新辅助化疗疗效及预后的关系

目的探讨血清肿瘤异常蛋白(TAP)、胃泌素释放肽前体(proGRP)、细胞角蛋白19片段(cyfra21-1)与Ⅲ~Ⅳ期非小细胞肺癌(NSCLC)新辅助化疗(NCT)疗效及预后的关系。方法选取100例Ⅲ~Ⅳ期NSCLC患者为研究对象,所有患者均采用NCT治疗,根据疗效将患者分为有效组和无效组,比较不同疗效患者化疗前后TAP、proGRP、cyfra21-1水平,分析化疗后TAP、proGRP、cyfra21-1水平对NSCLC患者NCT疗效的评估价值。所有患者均随访1年,分析化疗后TAP、proGRP、cyfra21-1表达水平与预后的关系。结果化疗后,有效组TAP、proGRP、cyfra21-1水平低于无效组(P<0.05)。ROC曲线结果显示,TAP评估NSCLC患者NCT疗效的AUC和截点值分别为0.739、178.18μm 2,proGRP评估NSCLC患者NCT疗效的AUC和截点值分别为0.810、52.21 ng/L,cyfra21-1评估NSCLC患者NCT疗效的AUC和截点值分别为0.775、7.70μmol/L,联合评估NSCLC患者NCT疗效的AUC为0.913,高于单项诊断(P<0.05)。TAP、proGRP、cyfra21-1高表达患者的1年生存率均低于低表达患者(P<0.05)。结论TAP、proGRP、cyfra21-1联合评估Ⅲ~Ⅳ期NSCLC患者NCT疗效具有较高价值,且其均与NSCLC患者预后密切相关。

减蛋综合征病毒pVⅢ基因和E3区序列测定及结构分析

克隆了减蛋综合征病毒（ＥＤＳＶ）中国分离株基因组ＨｉｎｄⅢ－Ｅ片段，并分析了其核苷酸序列。该片段位于基因组中央５８．７～６８．９物理图谱单位（ｍｕ），片段全长共３１９４ｂｐ。其中含有１个完整的和２个不完整的开放性读码框架（ＯＲＦ），分别编码ｐＶⅢ蛋白、１００Ｋ蛋白基因Ｃ端和纤维蛋白Ｎ末端部分。ｐＶⅢ蛋白基因由７５３个碱基（ｎｔ）组成，编码２５０个氨基酸（ａａ），编码产物的分子量为２８５００。氨基酸水平的同源性比较表明，ＥＤＳＶ的ｐＶⅢ蛋白与禽腺病毒１型（ＦＡＶ１）及人和其他动物腺病毒ｐＶⅢ蛋白的同源性为２８％～３６％，而与羊腺病毒（ＯＡＶ）的同源性高达５２％，提示ＥＤＳＶ与ＯＡＶ间的亲缘关系更近。按照人腺病毒基因组结构的特征，在ｐＶⅢ和纤维蛋白基因之间，应为Ｅ３区，但该序列只有２４５个ｎｔ组成，序列中保留了ＴＡＴＡＢｏｘ和ｐｏｌｙＡ信号。但除了２个仅编码４９ａａ和３２ａａ多肽的ＯＲＦｓ外，不编码任何产物，且核苷酸序列与其他腺病毒的Ｅ３区无同源性，证实此区无明显Ｅ３区样结构。

减蛋综合征病毒PVⅢ蛋白基因的克隆与序列分析

根据减蛋综合征病毒PV 蛋白基因序列设计了1对特异性引物,应用降落PCR法对PV 蛋白基因进行了扩增。将扩增产物克隆至pMD18-T载体中,经酶切和PCR鉴定,初步证明了目的片段的正确性。进一步核苷酸序列测定表明,所扩增的基因片段长度为0.756kb,共编码250个氨基酸。

减蛋综合征病毒PVⅢ蛋白基因的扩增克隆与鉴定

应用降落PCR法对减蛋综合征病毒PVⅢ蛋白基因进行了扩增 ,结果得到 1.1kb的扩增产物。将该扩增产物克隆至 pMD18-T载体中 ,经酶切和PCR鉴定后 ,证明了目的片段的正确性 ,从而实现了EDSV -gDNAPVⅢ基因的克隆 ,这不仅为进一步阐明EDSVPVⅢ结构与功能的关系打下了基础 ,而且为PVⅢ基因的表达及EDS基因工程苗的研究也奠定了良好的基础。

An Inner Membrane Protein(Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

Xanthomonas oryzae pv. oryzicola （Xoc） causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein （Imp）, showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R？imp was constructed in the wild-type RS105. The R？imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide （EPS） production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

An enriched environment increases the expression of fibronectin type Ⅲ domain-containing protein 5 and brain-derived neurotrophic factor in the cerebral cortex of the ischemic mouse brain

Many studies have shown that fibronectin type III domain-containing protein 5(FDNC5) and brain-derived neurotrophic factor(BDNF) play vital roles in plasticity after brain injury. An enriched environment refers to an environment that provides animals with multi-sensory stimulation and movement opportunities. An enriched environment has been shown to promote the regeneration of nerve cells, synapses, and blood vessels in the animal brain after cerebral ischemia;however, the exact mechanisms have not been clarified. This study aimed to determine whether an enriched environment could improve neurobehavioral functions after the experimental inducement of cerebral ischemia and whether neurobehavioral outcomes were associated with the expression of FDNC5 and BDNF. This study established ischemic mouse models using permanent middle cerebral artery occlusion(pMCAO) on the left side. On postoperative day 1, the mice were randomly assigned to either enriched environment or standard housing condition groups. Mice in the standard housing condition group were housed and fed under standard conditions. Mice in the enriched environment group were housed in a large cage, containing various toys, and fed with a standard diet. Sham-operated mice received the same procedure, but without artery occlusion, and were housed and fed under standard conditions. On postoperative days 7 and 14, a beam-walking test was used to assess coordination, balance, and spatial learning. On postoperative days 16–20, a Morris water maze test was used to assess spatial learning and memory. On postoperative day 15, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex were analyzed by western blot assay. The results showed that compared with the standard housing condition group, the motor balance and coordination functions(based on beam-walking test scores 7 and 14 days after operation), spatial learning abilities(based on the spatial learning scores from the Morris water maze test 16–19 days after operation), and memory abilities(based on the memory scores of the Morris water maze test 20 days after operation) of the enriched environment group improved significantly. In addition, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex increased in the enriched environment group compared with those in the standard housing condition group. Furthermore, the Pearson correlation coefficient showed that neurobehavioral functions were positively associated with the expression levels of FDNC5 and BDNF(r = 0.587 and r = 0.840, respectively). These findings suggest that an enriched environment upregulates FDNC5 protein expression in the ipsilateral cerebral cortex after cerebral ischemia, which then activates BDNF protein expression, improving neurological function. BDNF protein expression was positively correlated with improved neurological function. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, China(approval Nos. 20160858 A232, 20160860 A234) on February 24, 2016.

Effects of gentiana scabra bage on expression of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with liver fibrosis

Objective:To exploit- the effects of gentiana scabra bage on the expression of hepatic collagen proteins in Paragonimus skrjabini rats with liver fibrosis.Methods:Immunohistochemical technique was used to observe the changes of content of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with Liver fibrosis before and after the gentiana scabra bage treatmeat.Results:Comparing with the model group,changes of hepatic tvpe Ⅰ and type Ⅲ collagen proteins in gentiana scabra bage treated group were significantly weakened.Conclusions:Gentiana scabra bage treatment can reduce the content of hepatic type Ⅲ and type Ⅰ collagen protein significantly in Paragonimus skrjabini rats with liver fibrosis,thereby,playing the role against hepatic fibrosis.

Xoryp_08180 of Xanthomonas oryzae pv.oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein HY 322 Gene of Vibrio alginolyticus

In this study,Hy322 gene was cloned from Vibrio alginolyticus.The total length of its gene was 969 bp,and it could encode 322 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Hy322 of V.alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools.The results showed that Hy322 is an unstable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to α-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and V.harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.HY322 contains a FliN super family conserved domain associated with Flagellar motor switch.Bioinformatics analysis showed that the B-cell preponderant epitopes of Hy322 might be localized in the regions of 32-33,100-102,138-140,215-216,235-238 and 246-249.The 3D structure model of Hy322 subunit was simulated by SWISS-MODEL software and itwas found that the yscQ of Yersinia were similar and the similarity was 42.25%.In this study,the feasibility of Hy322 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.

钡A-偶氮氯膦Ⅲ络合物探针分光光度法测定蛋白质

在pH 1.0～ 1.5的酸性介质中 ,蛋白质与Ba 偶氮氯膦Ⅲ络合物发生作用 ,导致络合物溶液褪色 ,其最大吸收波长处吸光度的降低值与蛋白质的浓度成正比。基于此建立了以Ba 偶氮氯膦Ⅲ络合物为光谱探针 ,分光光度测定蛋白质含量的新方法。该法灵敏度高 ,选择性好。蛋白质 (以牛血清白蛋白为例 )的浓度在 0～ 4 0mg/L范围内服从比尔定律 ,表观摩尔吸光系数ε656=6 .4 7× 10 5L·mol-1·cm-1。生物体内常见物质均不产生干扰 ,直接应用于人血清样品中蛋白质总量的测定 。

瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达

采用刚果红染色法从瑞氏木霉cDNA文库中分离到一株具有CMCase活性的阳性克隆 ,测序结果显示该基因所编码的蛋白质为瑞氏木霉内切葡聚糖酶Ⅲ (EGⅢ )。对重组酿酒酵母所产生的EGⅢ进行了酶学性质分析 ,其最适pH为 5 0 ,最适温度为 60℃。检测了酿酒酵母蛋白质分泌系统组分SSO2和SEB1对EGⅢ分泌的影响。结果表明 ,在可过量表达蛋白质分泌系统组分SSO2的酿酒酵母H837中 ,EGⅢ的分泌量最高。由此分析 ,酿酒酵母SSO2蛋白可能在EGⅢ的分泌中 ,是一个限速步骤。通过PCR方法删除EGⅢ基因 5′端非翻译区的 98bp核苷酸序列使EGⅢ表达量提高了 5 3倍。这提示我们 ,瑞氏木霉的EGⅢ基因在酿酒酵母细胞中表达时 ,其mRNA 5′端先导序列中可能存在影响该基因表达水平的调控序列。

偶氮氯膦-Ⅲ分光光度法测定蛋白质

基于酸性介质中OP-100存在条件下蛋白质与偶氮氯膦-Ⅲ（CPA-Ⅲ）可迅速反应生成复合物（CPA-Ⅲ）-BSA,并产生特征吸收峰的特征,结合分光光度法精确检测蛋白质的质量浓度.结果表明：（CPA-Ⅲ）-BSA最大吸收波长为674nm;蛋白质质量浓度在0-300μg/mL内与吸光度差值（ΔA）呈良好的线性关系,其线性回归方程为ΔA=0.003 1ρ＋0.003 9（ρ：μg/mL）,相关系数r=0.996 4;检出限为1.55μg/mL.利用该方法测定人血清和鸡蛋清样品中蛋白质的质量浓度,相对标准偏差为2.43%-4.35%,加标回收率为97.97%-103.07%.

闹羊花素-Ⅲ对昆虫血淋巴能源物质含量的影响

用闹羊花素 -Ⅲ处理菜粉蝶 5龄幼虫后 ,观察试虫血淋巴总量及其蛋白质、总糖、甘油酯含量的变化。结果表明以每虫 1～ 5 μg处理后 6 0h ,血淋巴总量和蛋白质含量均降低 4 0 %～ 5 0 % ,总糖含量降低 14 %～30 % ,甘油酯含量可降低 77%。

血浆蛋白C、蛋白S、抗凝血酶Ⅲ活性与脑梗死的相关性研究

目的研究血浆蛋白C(PC)、蛋白S(PS)和抗凝血酶Ⅲ(AT-Ⅲ)活性动态变化与脑梗死发病的关系。方法对51例急性期脑梗死患者、23例恢复期脑梗死患者及20例正常对照组进行血浆PC、PS和AT-Ⅲ的活性进行检测,并分析其与脑梗死发病时间、梗死范围和发病年龄之间的关系。结果急性期PC、PS和AT-Ⅲ活性明显低于恢复期和对照组(P<0.05);恢复期PC活性与对照组相比无明显差异(P>0.05);而恢复期PS和AT-Ⅲ活性与对照组相比存在显著差异(P<0.05)。大梗死组和中梗死组中的PC、PS和AT-Ⅲ活性明显低于小梗死组和对照组(P<0.05);而大梗死组和中梗死组相比以及小梗死组与对照组相比均无明显差异(P>0.05)。进一步比较发现,≤45岁的脑梗死组的PC、PS和AT-Ⅲ活性明显低于>45岁组(P<0.05)。结论 PC、PS和AT-Ⅲ活性降低是脑梗死发生的重要因素,也与45岁以下青年人脑梗死的发生有一定的相关性。

抗凝血酶Ⅲ、蛋白C、蛋白S活性与脑梗死的相关性研究

目的研究血浆抗凝血酶Ⅲ(AT-Ⅲ)、蛋白C(PC)和蛋白S(PS)活性动态变化与脑梗死发病的关系。方法对126例脑梗死患者按年龄、发病时间和梗死范围分组,同时选择健康体检30例作为健康对照组,检测血浆中AT-Ⅲ、PC和PS的活性,并分析其活性变化与脑梗死年龄、发病时间及梗死范围之间的关系。结果急性期青年组的AT-Ⅲ、PC、PS活性低于急性期中老年组、差异有统计学意义(P<0.05);急性期AT-Ⅲ、PC、PS活性低于恢复期和对照组,差异有统计学意义(P<0.05);恢复期AT-Ⅲ和PS活性低于对照组,差异有统计学意义(P<0.05),而恢复期PC活性与对照组比较,差异无统计学意义(P>0.05);进一步研究发现,AT-Ⅲ、PC、PS活性在小梗死组、中梗死组、大梗死组间呈逐渐降低的趋势,差异均有统计学意义(P<0.05)。结论AT-Ⅲ、PC、PS活性降低是脑梗死发生的重要因素,特别与45岁以下青年人脑梗死的发生密切相关。观察其活性变化对判断脑梗死病情的发展有重要的临床意义。

红外光谱酰胺Ⅲ带用于蛋白质二级结构的测定研究

用甲醇对 BSA和 Rase A等蛋白质进行变性处理 ,结合蛋白质酰胺 带的拟合结果对酰胺 带各二级结构的谱峰进行了初步指认 :1 330～ 1 2 90 cm- 1为 α-螺旋 ;1 2 95～ 1 2 6 5 cm- 1为 β-转角 ;1 2 70～ 1 2 4 5cm- 1 为无规卷曲 ;1 2 5 0～ 1 2 2 0 cm- 1 为β-折叠 .依据这些谱峰归属 ,对一些已知二级结构的蛋白质进行了测定 ,所得结果与 X射线衍射数据以及酰胺 带的定量结果基本一致 .

钇(Ⅲ)-偶氮胂Ⅲ配合物探针分光光度法测定蛋白质的研究

在pH2．3～2．5的B-R缓冲介质中，蛋白质与钇（Ⅲ）偶氮胂Ⅲ配合物发生结合反应，引起配合物溶液褪色及吸光度降低。在一定范围内其最大吸光度降低值与蛋白质的浓度成正比。基于此，建立了以钇（Ⅲ）-偶氮胂Ⅲ配合物为光谱探针，分光光度测定蛋白质含量的新方法。该法灵敏度高，线性关系好，人血清白蛋白的含量在0～20mg／L范围内与配合物吸光度的降低值呈现良好的线性关系；表观摩尔吸光系数ε652为2．60×10^6L·mol^-1·cm^-1；生物体内的常见物质基本上不干扰测定。本法可直接应用于人血清样品中蛋白质总量的测定。

苦参碱对肺纤维化大鼠核转录因子-κB及胶原蛋白Ⅲ的影响

目的通过观察苦参碱干预肺纤维化(PF)大鼠核转录因子-κB(NF-κB)及胶原蛋白Ⅲ的变化,探讨苦参碱的抗PF机制。方法50只SD雄性大鼠随机分为对照组、模型组、高苦参碱组、中苦参碱组和低苦参碱组,每组10只。对照组每日腹腔注射生理盐水,共30 d;模型组和高、中、低苦参碱组均每日腹腔注射博莱霉素7.5 mg.kg-1,10 d后模型组每日腹腔注射生理盐水1 mg.kg-1,高、中、低苦参碱组每日分别腹腔注射苦参碱100 mg.kg-1、80 mg.kg-1、50 mg.kg-1,共20 d。观察各组肺组织形态、NF-κB及胶原蛋白Ⅲ在肺组织的表达变化。结果模型组和高、中、低苦参碱组肺组织呈纤维化表现,模型组纤维化程度明显高于对照组(P<0.05);高、中苦参碱组与模型组相比纤维化程度减轻(P<0.05);低苦参碱组与模型组相比纤维化程度差异无统计学意义(P>0.05)。模型组NF-κB、胶原蛋白Ⅲ的表达均高于对照组,差别有统计学意义(P<0.01);高、中苦参碱组NF-κB、胶原蛋白Ⅲ的表达均较模型组减少(P<0.01);低苦参碱组NF-κB、胶原蛋白Ⅲ表达与模型组相比无统计学意义(P>0.05)。高、中、低苦参碱组组间两两比较,NF-κB、胶原蛋白Ⅲ均有统计学意义(P<0.01)。结论苦参碱可能通过减少NF-κB、胶原蛋白Ⅲ的表达,减轻大鼠PF程度。

偶氮胂Ⅲ-镧(Ⅲ)褪色光度法测定蛋白质

在pH 2.5的酸性介质中, 蛋白质与偶氮胂Ⅲ-镧（Ⅲ）络合物能够相互作用, 形成超分子复合物而使溶液褪色. 最佳条件下, 在650 nm处吸光度的降低值, 与加入的牛血清白蛋白（BSA）或人血清白蛋白（HSA）、溶菌酶（Lyso）质量浓度在一定范围内呈线性关系. BSA在0.5～35 mg/L、 HAS在1～40 mg/L, 溶菌酶（Lyso） 1.7～45 mg/L, 相对标准偏差为0.82% （n=7）. 方法用于人血清总蛋白量的测定.