酶蛋白

①Rat MSC and VSMC were cultured and identified, respectively. MSC were labeled with DAPI firstly, and then co-cultivated with VSMC. The changes of morphology and ultrastructure of co-cultured cells were observed. Immunfluorescence analysis was performed by using monoclonal antibodies against specific antigen.②We established the regulatable system in two steps: a stable MSC line expressing rtTA has been constructed and characterized firstly by transfected with pUHD 17-1hyg and then selected by hygromycin B; in a second step, this line was used for trandfer the AT2R gene to MSC to get the well establishing double stable MSC lines;③The expression of AT2R regulated by doxycycline was evaluated by western blot;④The MSCs were transduced into rat carotid arteries with regulatable AT2R gene after the establishment of rat carotid balloon injury restenosis model. The intimal/medial area ratio were measured by digital analysis system.

研究方法：(1)密度梯度离心法及胶原酶消化法分别培养原代大鼠MSC及VSMC，细胞共培养并行免疫荧光化学染色和透射电镜观察超微结构；(2)组成受Dox调控的哺乳动物表达系统的四种成分的转化、扩增及提纯并酶切鉴定；(3)采用常规分子生物学方法连续两个回合转染体外培养的MSC，并分别采用发光计检测不同细胞克隆萤光素酶活性改变以及RT-PCR方法检测AT2R目的基因mRNA表达情况，根据各个细胞克隆受Dox调控表达的程度，选择低背景、高诱导表达AT2R的细胞系，作为双重稳定MSC细胞系；蛋白免疫印迹法观察该细胞系在Dox调控下AT2R表达的时相性、持续性及在不同浓度Dox调控下的表达情况；(4)建立大鼠颈动脉球囊损伤动物模型，将双重稳定MSC在术中导入血管，分别于14 d、28 d进行病理切片，检测可调控AT2R对新生内膜增生的影响；采用RT-PCR免疫组织化学免疫荧光等技术观察AT2R基因在新生内膜中的表达以及细胞外基质成分表达的改变，TUNEL法检测血管组织中细胞凋亡的变化情况。

The DNA of Yak PrP expressed plasmid extracted from the recombinant expressed bacterium was identified by PCR amplification and two-ezyme digestion. The expressed product was obtained from the recombinant bacterium inducted under the optimized expressed condition (37℃, 1 mmol/L IPTG, 6 h), and it was determined by SDS-PAGE and Western blotting. The GST-BoPrP(23—242) fusion proteins were collected by two ways: the first is to purify and renature from the inclusion bodies; the second is to separate by SDS-PAGE.

将保存的含有牦牛PrP基因重组表达菌提取质粒DNA，进行PCR扩增和双酶切鉴定，并在优化诱导表达条件(37℃，1mmol/L IPTG，6h)下，获得的表达产物进行SDS-PAGE和Western blotting检测；将鉴定之后的重组表达菌诱导表达后，通过两种方法回收GST-BoPrP(23～242)融合蛋白：其一，是从包涵体中提取和复性GST-BoPrP(23～242)融合蛋白；其二，是通过SDS-PAGE直接分离并纯化GST-BoPrP(23～242)融合蛋白。

METHODS:The gene of human ET1 was synthesized according to the preferential codons of E. coli, cloned into the EcoRI and SalI sites of vector pThioHisA. The recombinant plasmid pThioHisA-ET1 was constructed , sequenced and transformed into E.coli TOP10. Induced and expressed fusion protein were identified and analysed by 12% SDS-PAGE and densitometry analyses. After the elution, denature and renature, the fusion protein Thioredoxin-ET1 was obtained by ProBondTM chromatogragraphy. The purity of Thioredoxin-ET1 was detected by HPLC. Inoculate Thioredoxin-ET1 once per mouse every 2 weeks in 25, 50 and 100μg separately on 3 groups for 4 times. 10 days after last inoculation, we obtained venipuncture blood.

根据人ET1的多肽序列合成ET1基因，将其插入到pThioHisA的EcoRI和 SalI位点，重组质粒pThioHisA-ET1进行酶切鉴定及序列测定验证后转化TOP10，IPTG诱导的重组菌经SDS-PAGE检测融合蛋白Thioredoxin-ET1的表达量；表达的融合蛋白用ProBond亲合层析纯化并经HPLC鉴测其纯度；每只小鼠按25、50、100ug/次剂量的Thioredoxin-ET1每两周免疫一次，共4次，最后一次免疫10d后制备抗血清，经Western blot和ELISA检测证明Thioredoxin-ET1融合蛋白具有ET1免疫反应原性。

Objective To present a new serological method for diagnosis of breast cancer. Methods Seral anti-P 53 antibody was detected with immuno-PCR.The expression of P 53 in tissue was detected with enzyme imunohisto-chemistry technique.

目的 探讨乳腺癌患者P 53 蛋白及抗体表达情况方法采用免疫PCR方法检测血清抗P 53 蛋白抗体，酶免疫组化方法检测组织P 53 蛋白表达。

The expression changes of ICAM-1 and p65 mRNA level were detected by reverse transcription-polymerase chain reaction and the expression of p65 protein in AR42J cell lines were detected with Stept Avidin-Biotin complex respectively, and the changes of amylase in culture medium with chromatometry.

逆转录－多聚酶链反应半定量法观察细胞间黏附分子(ICAM-1)和p65亚单位mRNA表达的变化；链酶亲和素－生物素－过氧化物酶复合物法检测p65蛋白在AR42J细胞中的表达；人工碘比色法观察培养液上清中淀粉酶的活性改变。

The expression changes of TNF-α and p65 mRNA expression were detected by reverse transcription-polymerase chain reaction, the TNF-α level in the supernatant was determined by ELISA, and the expression of p65 in AR42J cell lines was detected by Stept Avidin-Biotio Complex.

逆转录－多聚酶链反应半定量法观察肿瘤坏死因子/p65亚单位mRNA表达的变化；ELISA法检测TNF-α在细胞上清中质量浓度的变化；链酶亲和素－生物素－过氧化物酶复合物法检测p65蛋白在AR42J细胞中的表达。

NodD binds to and bends target promoters through anchoring two tandem and individual specific DNA sites. NodD functions as a tetramer, which has a V-shaped main body. Tetrameric NodD is to change its own conformation rather than its oligomeric forms in response to small signal molecules. The specific interaction between each NodD DNA-binding domain and each specific DNA site does not alter itself in spite of naringenin induction, and the induced conformational change is transferred from protein to DNA. Only the DNA conformation incited by induced NodD is competent for RNA polymerase to form the transcriptional open complex. It cannot be excluded that NodD may have protein-to-protein contacts with RNA polymerase, and that the NodD conformational change may also directly contribute to the transcriptional open complex formation. However, the NodD conformational change itself cannot serve as the determinant of the transcriptional molecular switch.

通过研究，我们提出了初步的NodD操纵子激活模型：第一，四聚体是NodD蛋白的功能单位，它通过铆钉两个串联的相对独立的DNA靶位点结合被诱导的启动子；第二，小分子配基的结合是改变NodD四聚体的构象而不是引发不同形式的寡聚体，在我们的模型中，NodD四聚体缩小其V形主体的弯折角，进而缩短其DNA结合功能域的间距；第三，小分子信号的诱导并没有改变NodD的DNA结合域和其DNA靶位点的相互作用，NodD的构象改变由蛋白质经其双铆钉位点传递给DNA；第四，只有诱导状态的NodD激发的DNA构象才能有效地使RNA聚合酶形成转录开放复合物；第五，不排除NodD与RNA聚合酶可能有直接的相互接触位点，不排除NodD构象的改变可能直接有利于RNA聚合酶形成转录开放复合物，但是我们认为NodD构象改变本身不是充当转录激活开关的决定因素。

Therefore,a sequence specific artificial zinc finger proteinis created when replacing the amino acids in the specific sites of C2H2 zinc finger protein and then fusing the protein to other activation or repression domains,while remaining the backbone of the zinc finger protein unchange...

ZFP可以介导靶基因的转录调控，抑制或激活特定基因的表达与配体依赖的靶基因激活或抑制；对DNA进行修饰，如人造限制性内切酶，重组酶，整合酶；抗病毒感染等。因此，人造锌指蛋白应用前景广阔，研究价值显著，是未来人类基因治疗的革命性的工具。

It is caused by mutations in dyskerin-encoding genes, telomerase-encoding RNA component genes and reverse transcriptase genes, as well as other uncharacterized genes. There are three inherited forms, including X-linked, autosomal dominant and autosomal recessive inheritance.

先天性角化不良是与编码角化不良蛋白基因、编码端粒酶的RNA组份基因、编码端粒酶的逆转录酶基因突变及其他未确认的致病基因突变引起的基因病，其遗传方式有：X-性联隐性遗传、常染色体显性遗传及常染色体隐性遗传。

Effects of microbial transglutaminase on the tensile properties of plastic made from soy protein isolates are studied. The results show that the tensile strength and Young modulus of the protein plastic are increased from 9.2MPa and 265.4MPa of the control, which is not treated with TG, to 14.7MPa and 390.1Mpa respective, which is treated with TG at 50℃ for 60min. High molecular weight protein conjugates are formed with TG treatment, and they are responsible for the enhanced tensile properties, denaturalizing temperature and enthalpy values.

研究了微生物谷氨酰氨转胺酶对大豆分离蛋白塑料拉伸性能的影响，发现在50℃时转氨酶对大豆分离蛋白催化聚合反应60 min，形成的高分子聚合物使大豆分离蛋白的抗拉强度和杨氏模量分别由未经处理时的9.2 MPa和265.4 MPa增加到14.7 MPa和390.1 MPa，同时增加了蛋白质的变性温度和热焓值。

This one mode pays close attention to network credence foundation of the businessman very much.

这一模式非常关注商人的网络信用基础。

Cell morphology of bacterial ghost of Pasteurella multocida was observed by scanning electron microscopy and inactivation ratio was estimated by CFU analysi.

扫描电镜观察多杀性巴氏杆菌细菌幽灵和菌落形成单位评价遗传灭活率。