本试验成功构建了香蕉苞片花叶病毒(Banana bract mosaic virus,BBrMV)外壳蛋白(coat pro-tein,CP)基因的原核表达载体,并诱导表达了34 kDa的融合蛋白His.CP。对该原核表达蛋白的可溶性分析表明,该融合蛋白以包涵体形式存在。利用组氨酸标签纯化试剂盒对目的蛋白进行了纯化,获得了高纯度的融合蛋白。以纯化的蛋白为抗原免疫健康家兔,成功制备了抗BBrMV CP基因编码蛋白的兔抗血清。Western-blotting结果表明这种抗血清有很强的特异性。血清效价测定的效价在51 200倍以上,对植物材料的合适检测浓度为1:800～1:3 200。

Banana bract mosaic virus (BBrMV) coat protein (CP) gene was cloned, and prokaryotic expression recombinant plasmid pET- CP was constructed by inserting the cloned gene into pET -28b ( + ). Induced by IPTG,E. coli BL21 (DE3) containing pET- CP produced fusion proteins about 34 kDa in size. Soluble analysis of the fusion protein indicated that it was in the inclusion body. The highly purified interest protein was obtained by using the histidine labeling of N - terminus of the protein. The special antibody was generated to the protein through the purified protein immunizing healthy rabbit. Indirect enzyme immunoassay suggested antibody titre was higher than 1:51 200. The available antibody concentration for virus detection from plant material was 1:800 - 1 : 3 200.

从海洋球石藻Emiliania huxleyi病毒EhV99B1的基因组中克隆了丝氨酸蛋白酶(Sp)基因(GenBank登录号:KC161207),对该基因的开放阅读框(ORF)进行系统的生物信息学分析,并在大肠杆菌中融合表达,通过亲和层析法获得了纯化的重组Sp。结果表明:EhV99B1-Sp基因的ORF为1 110bp,编码368个氨基酸,蛋白相对分子质量为39.5kDa;该基因片段与GenBank中EhV86-Sp的同源性很高,核苷酸及其对应的氨基酸序列同源性分别为95%和97%,而与其他物种Sp序列的同源性仅为28%~32%,说明其可能是丝氨酸蛋白酶家族中的一个新成员;预测的二级结构特征显示EhV99B1-Sp的蛋白结构域中具有典型的LTAGHC(组氨酸活性位点区域)和AICNGDSGGPLF(丝氨酸活性位点区域)两个丝氨酸蛋白酶催化活性位点的氨基酸基序,是一个两次跨膜蛋白;将该基因在大肠杆菌中进行低温诱导表达,得到分子量为60kDa的重组蛋白,经鲤鱼肌肉丝氨酸蛋白酶(MBSP)抗体检测证实为Sp,且重组蛋白在大肠杆菌细胞中具有明显的生物学活性。本研究结果为进一步探讨EhV99B1-Sp在病毒与宿主相互作用过程中的调节作用及其功能与应用奠定基础。

A serine protease (Sp)gene (GenBank accession number:KC161207)was cloned from the genome of marine microalgal Emiliania huxleyi virus (Coccolithovirus )EhV99B1 isolate.Bioinformatic analysis was pre-formed on its open reading frame and the recombinant protein was expressed in E.coli and purified by affinity chromatography.Results showed that the length of the ORF of EhV99B1-Sp was 1 110 bp,which encoded a pro-tein of 386 amino acids with a molecular mass of 39.5 kDa and pI of 6.255.EhV99B1-Sp shared a high sequence similarity with EhV86-Sp,between whom the similarities of nucleotide sequences and deduced amino acid sequences were 95% and 97%,respectively.However,the sequence similarity between EhV99B1-Sp and serine protease from other organisms was only 28% to 32%.Sequences analysis suggested that EhV99B1-Sp might be a new member of the serine protease family.Secondary structure prediction showed that EhV99B1-Sp protein contained two motifs of typical serine protease catalytic active s

目的人博卡病毒(HBoV)是近几年发现的一种导致人支气管炎的病原体,本研究旨在表达纯化其VP1衣壳蛋白独有区(VP1-U)抗原,为免疫学诊断治疗奠定基础。方法优化HBoV VP1-U DNA密码子,以适应大肠杆菌原核表达密码子亲嗜性。将密码子优化改造的DNA序列克隆到pET30a(+)原核表达载体,C末端融合表达组氨酸亲和纯化标签。LB培养基增菌,IPTG诱导表达。采用自行设计的纯化缓冲液体系,镍株亲和纯化,超滤浓缩。结果VP1-U蛋白表达效率约为50%,纯化活性蛋白浓度为3.3mg/ml。结论经过密码子优化,合理设计工艺流程,原核表达系统可以高效表达纯化VP1-U活性蛋白抗原。

Objective To express and purificate of HBoV VP1-U protein antigen.Methods HBoV VP1-U codons was Optimized to accommodate E.coil codon tropism,and The DNA sequence which fused to an C-terminal (His)6-tagged that allowed single-step isolation by Ni2+ affinity chromatography,was cloned into pET30a(+)vector.The re-combinant plasmid was transformed into E.coli strain BL21(DE3)induced expression by IPTG.Extracts protein from cell was loaded onto Ni2+ affinity column and (His)6-tagged proteins are selectively eluted with self-designed buffers system.Results The VP1-U expression efficiency was approximately 50%,the concentration of the purified active pro-tein was about 3.3 mg/mL.Conclusion By codons optimization and rational process,VP1-U active protein antigen can be efficiently expressed and purified in E.coil prokaryotic expression systems.

为了克隆猪输血传播病毒2型（TTV2）ORF1基因，构建原核表达载体，在大肠杆菌BL21（DE3）中诱导表达猪TTV2 ORF1蛋白，并对其表达条件进行优化。利用普通PCR从猪TTV2阳性样本中扩增出TTV2 ORF1基因，利用分子克隆的方法将猪TTV2 ORF1基因克隆到原核表达载体pcoldI上，构建表达载体pcold-ORF1，在大肠杆菌中诱导表达带有6个组氨酸标签的ORF1融合蛋白。并对影响重组蛋白表达的3个因素，即诱导时间、诱导温度和IPTG浓度进行优化，确定了pcold-ORF1重组蛋白表达的最佳表达条件。 SDS-PAGE分析结果表明，重组蛋白在BL21中高效表达，以包涵体形式表达为主，分子质量约为39 kDa。蛋白表达量随诱导时间增加而有所增加，在15℃时表达量最高，而IPTG浓度对蛋白的表达量没有显著影响。 Western Blotting 结果表明，重组蛋白与猪TTV2阳性血清特异性反应，具有很好的免疫原性。成功获得TTV2-ORF1基因，构建了原核表达载体并获得高效表达，为TTV的ELISA检测方法的建立提供抗原。且重组蛋白在15℃条件下，加入0．2 mmol/L浓度的IPTG，诱导24 h，表达条件最佳。

To clone the open reading frame 1(ORF1)gene of porcine torque teno virus type 2 and construct a prokaryotic expression vector .TTV2 ORF1 recombinant protein was expressed in Escherichia coli and optimal ex-pression was performed.The ORF1 gene of TTV2 was amplified from the TTV2 positive samples and sub-cloned in a prokaryotic expression vector pcoldI .The constructed recombinant plasmid pcold-ORF1 was transformed to E.coli BL21 for expression the ORF1 fusion protein with six histidine-tag under induction of IPTG .The induction time ,in-duction in different temperature and IPTG concentrations were optimized .The recombinant fusion protein had high expression level in BL21(DE3),and SDS-PAGE analysis showed the exogenous gene was mainly expressed as in-clusion bodies and its molecular weight was about 39 kDa.The induction time were positively related trends with re-combinant protein expression ,the best induction temperature was 15 ℃,whereas IPTG concentration had no signifi-To clone the

目的 通过膜性肾病经典的动物模型被动Heymann肾炎(PHN),探讨足细胞自噬在膜性肾病病变过程中的作用,以及自噬激活可能的细胞内机制.方法 SPF级健康雄性SD大鼠按随机数字表法分为5组即对照组、PHN 2 d组、PHN 4 d组、PHN 7 d组、PHN 21 d组,建立大鼠PHN模型.采用透射电镜观察足细胞形态和自噬泡,Weibel-Gomez点计数法计数单个肾小球足细胞数量,免疫组织化学染色检测肾小球内膜攻击复合物C5b-9的沉积与半胱氨酸天冬氨酸蛋白酶(caspase)-3的表达,原位缺口末端标记(TUNEL)法检测肾小球内细胞凋亡,Western印迹法检测肾小球自噬相关蛋白微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)、葡萄糖调节蛋白(GRP)78、磷酸化蛋白激酶R样内质网激酶(phosphorylated protein kinase R-like ER kinase,p-PERK)、磷酸化c-Jun氨基末端激酶(phosphorylated c-jun aminoterminal kinase,p-JNK)、活化转录因子6α(activating transcription factor 6α,ATF6α)的表达.结果 (1)造模后第4天起即可在肾小球内观察到C5b-9沿基底膜呈线状沉积.同时,透射电镜下可见上皮下少许颗粒状电子致密物沉积及足突融合,病变随时间呈加重趋势,至第21天可见典型膜性肾病改变.此外,尿蛋白定量结果显示,造模第3天起尿蛋白量即有明显升高,至第21天时可高达(50.6±6.0) mg/24 h.(2)造模后第21天单位肾小球足细胞绝对数目较对照组显著下降(P＜0.05).(3) caspase-3表达和TUNEL染色结果一致显示,造模后第21天肾小球内可检出凋亡的足细胞.(4)透射电镜和自噬标志LC3Ⅱ的检测结果均显示,造模后第7天和第21天足细胞的自噬水平均显著升高,但第21天LC3Ⅱ的表达较第7天有所回落.(5)造模后第2天即可检出GRP78高表达,至第7天时显著升高,而病变后期(第21天)又有所回落;同时,非折叠蛋白反应传感器下游的3条信号通路(ATF6α、p-PERK及p-JNK)也在造模早期活化,后期(第21天)同样有所回落.结论 膜性肾病

Objective To investigate the role of autophagy in podocyte damage,and the intracellular mechanism of autophagy activation through passive Heymann nephritis (PHN) animal model.Methods Male Sprague-Dawley rats (n=40) were studied on day 0,2,4,7,and 21 after induction of PHN by injection of anti-Fx1A.Podocyte morphology and autophagosomes were observed by transmission electron microscopy.Podocyte numerical density was estimated by Weibel-Gomez =method.Cell apoptosis was detected by TUNEL assay and caspase-3 immunohistochemical staining.Expressions of autophagy markers and endoplasmic reticulum stress (ERS)-associated proteins were analyzed by Western blotting.Results (1) In PHN rats,immunohistochemical staining showed that C5b-9 deposited along glomerular basement membrane on day 4 to day 21.Small subepithelial electron -dense deposits and a part of foot process fusion were detected in the glomerulus of PHN rats on day 4 by transmission electron microscope,and podocyte damage was aggravat