艰难梭菌毒素B2型蛋白重组表达及活性分析

目的 构建艰难梭菌高毒力株2型毒素B(TcdB2)的枯草芽胞杆菌工程株,获得高效表达的生物活性TcdB2蛋白。方法 以ST1/RT027型菌株基因组DNA为模板,通过PCR扩增获得TcdB2全长基因片段,构建到PHT01载体中,转化枯草芽胞杆菌WB800N菌株,进行原核表达获得TcdB2全长蛋白,进一步通过细胞毒性试验验证重组TcdB2的生物活性。结果 成功构建了高效表达TcdB2的枯草芽胞杆菌工程株,并获得具有细胞毒性的重组蛋白TcdB2。结论 具有生物活性的重组TcdB2蛋白,为进一步研究艰难梭菌高毒力株的致病机制和作为疫苗候选靶标等奠定了基础。

超声血流定量参数结合BRAF对桥本甲状腺炎合并甲状腺癌的诊断价值

目的分析超声血流定量参数结合鼠类肉瘤滤过性毒菌致癌同源体B(BRAF)基因突变检测对桥本甲状腺炎(HT)合并甲状腺癌的诊断价值。方法以2020年6月至2022年6月于南京医科大学附属逸夫医院就诊的HT合并甲状腺结节患者86例为研究对象,依据病理检查结果,将合并甲状腺癌的患者为恶性组(n=26),将合并甲状腺良性结节患者为良性组(n=60)。恶性组患者中依据淋巴结转移情况分为转移组(n=10)和未转移组(n=16)。所有研究对象均接受彩色多普勒超声检查及BRAF基因突变检测,对比良、恶性组患者搏动指数(PI)、阻力指数(RI)、血流频谱收缩期峰值速度(S)、舒张末期血流速度(D)、收缩期峰值速度与舒张末期血流速度比值(S/D)、BRAF基因突变情况;对比转移组与未转移组患者彩色多普勒血流显像(CDFI)血流检出率、2~3级血流信号检出率及BRAF基因突变情况,以受试者工作特征(ROC)曲线评价上述血流参数与BRAF基因突变对HT合并甲状腺癌的诊断价值,并评估上述指标及BRAF基因突变对甲状腺癌淋巴结转移的诊断价值。结果恶性组患者的PI、RI、S、D、S/D及BRAF基因突变阳性率高于良性组(P<0.05);转移组患者的CDFI血流检出率、2~3级血流信号检出率及BRAF基因突变阳性率高于未转移组(P<0.05)。ROC曲线分析结果显示,RI、PI、S、D、S/D及BRAF基因突变联合评估HT合并甲状腺癌的曲线下面积(AUC)为0.951,敏感度为88.46%,特异度为93.33%,联合评估效能优于各指标单独评估;同时CDFI血流检出率、2~3级血流信号检出率及BRAF基因突变联合评估甲状腺癌淋巴结转移的AUC为0.938,联合评估效能优于各指标单独评估。结论超声血流定量参数结合BRAF基因突变对HT合并甲状腺癌及淋巴结转移均具有一定的评估价值,且各指标联合评估效能更佳。

Chloramphenicol improved expression of recombinant cholera toxin B subunit in Escherichia coli and its adjuvanticity

Background Cholera toxin B subunit （CTB） was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine. Methods Wild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ （IFN-γ） enzyme-linked immunospot （Elispot） assay was used to read out the specific T-cell immunity. Results Chloramphenicol was essential for the efficient expression of recombinant CTB （rCTB） in pET-30a/BL21 （DE3） system and could be optimized at the concentration of 0.625 μg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group （（734±240） spot forming cells/106 splenocytes） was higher than that induced by non-adjuvanted （（520±150） spot forming cells/10e splenocytes）, all responses against different antigens were enhanced in parallel. Conclusion CTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancincl the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.

CLINICAL APPLICATION OF BOTULINUM TOXIN TYPE B IN MOVEMENT DISORDERS AND AUTONOMIC SYMPTOMS

Objective To evaluate efficacy and safety of botulinum toxin type B (BTX-B) in treatment of movement disorders including blepharospasm, oromandibular dystonia, hemifacial spasm, tremor, tics, and hypersecretory disorders such as sia-lorrhea and hyperhidrosis. Methods A retrospective study of BTX-B injections in treatment of 58 patients with various neurological disorders was performed. The mean follow-up time was 0.9 ± 0.8 years. Results of the first and last treatment of patients with at least 3 injection sessions were compared. Results The response of 58 patients to a total of 157 BTX-B treatment sessions was analyzed. Of the 157 treatment sessions, 120 sessions (76.4%) resulted in moderate or marked improvement while 17 sessions (10.8%) had no response. The clinical benefits after BTX-B treatment lasted an average of 14 weeks. Of the 41 patients with at least 3 injection ses-sions (mean 10 ± 8.6), most patients needed increased dosage upon the last session compared to the first session. Nineteen patients (32.8%) with 27 sessions (17.2%) reported adverse effects with BTX-B treatment. Conclusions Though most patients require increased dosage to maintain effective response after repeated injections, BTX-B is an effective and safe treatment drug for a variety of movement disorders, as well as drooling and hyperhidrosis.

Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis

Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3-7 days after injection into the rat gastrocnemius muscle

Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

基于类器官培养的艰难梭菌毒素B损伤结肠上皮机制的初步研究

目的 建立艰难梭菌感染结肠类器官模型,对艰难梭菌毒素B(TcdB)损伤小鼠结肠上皮的作用及可能机制进行初步研究。方法 处死6~8周龄C57BL/6小鼠,取结肠标本,分离、收集结肠隐窝,以基质胶包埋后加入培养基,显微镜下观察记录生长情况;在巨大芽孢杆菌中表达重组艰难梭菌毒素B(rTcdB)蛋白,经镍柱纯化后加入结肠类器官体系继续培养,观察类器官生长情况并检测干性基因表达量变化,转录组测序分析。结果 5pmol/L rTcdB作用12 h即可致结肠类器官生长缓慢及部分死亡,干性基因Lgr5、Axin2、Bmi1表达较对照组显著上调。RNA-seq测序结果显示毒素作用组较对照组差异基因有3 140个,其中上调基因有1 936个,下调基因有1 204个。Gene Ontology富集分析显示损伤组差异基因主要富集的生物过程通路分别有低氧应答和对β干扰素、γ干扰素的细胞应答。同时蛋白质互作网络(Protein-Protein Interaction Networks, PPI)筛选出9个关键基因,其中的IFIT3、IFIT1、GBP3和GBP2的编码基因也参与了α/β干扰素、γ干扰素的细胞应答通路。京都基因与基因组百科全书(KEGG)通路富集分析结果表明有47条通路差异显著富集,包括花生四烯酸代谢通路和Wnt信号通路等。结论 TcdB可能通过调节干性基因表达,增强低氧应答通路,减弱α/β干扰素、γ干扰素的细胞应答通路,调节花生四烯酸代谢通路和Wnt信号通路等介导损伤结肠上皮细胞。

Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

霍乱毒素B亚基与草鱼呼肠孤病毒VP7融合基因的合成和原核表达

草鱼呼肠孤病毒(grass carp reovirus, GCRV)是一种致病力强,严重危害水产养殖业健康发展的病毒。免疫接种疫苗是预防GCRV的有效途径,而口服免疫接种对生活在水中的鱼类是一种理想的接种方式。GCRV衣壳蛋白VP7具有较好的免疫原性。霍乱毒素B亚基(cholera toxin B subunit, CTB)是一种较好的黏膜佐剂。本研究采用PCR技术,将CTB基因和VP7基因进行柔性融合,获得1260 bp的CTB-VP7融合基因,构建重组表达质粒pET28a-CTB-VP7,转化大肠杆菌BL21 (DE3),获得了表达CTB-VP7融合蛋白的菌株。当采用异丙基硫代半乳糖苷(IPTG)诱导时,获得CTB-VP7融合蛋白的分子量约49 kDa,与预期的分子量大小一致。当IPTG浓度为0.08 mmol·L^(-1),诱导温度为37℃,诱导转速为100 r·min~(-1),诱导表达5.0 h时,CTB-VP7融合蛋白的表达量最大,达到2.45 mg·L^(-1)。CTB-VP7融合蛋白在菌体中主要以包涵体的形式存在。在融合蛋白纯化时,洗脱缓冲液中咪唑浓度为200 mmol·L^(-1),目标蛋白纯度达96%。本研究通过基因合成和原核表达得到CTB-VP7融合蛋白,为进一步验证CTB-VP7融合蛋白能否作为抵抗GCRV的粘膜疫苗奠定了基础。

Expression of Shiga Toxin B Subunit at Cell Surface in E. coli K-12

The three parts(Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the Lamb fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.

清心抗炎饮治疗小儿急性柯萨奇B病毒性心肌炎邪毒侵心证临床研究

目的:观察清心抗炎饮治疗小儿急性柯萨奇B病毒性心肌炎邪毒侵心证的效果。方法:86例按随机数字表法分为两组各43例,对照组给予西药治疗,观察组给予清心抗炎饮治疗。结果:观察组总有效率高于对照组(P<0.05)。治疗后两组证候积分均降低(P<0.05),观察组降低更显著(P<0.05)。治疗后两组血清CK-MB、LDH、CPK水平均下降(P<0.05),且观察组低于对照组(P<0.05)。治疗后两组血清TNF-α、IL-6水平均下降(P<0.05),且观察组低于对照组(P<0.05)。结论:清心抗炎饮治疗急性病毒性心肌炎邪毒侵心证疗效较好。

Diphtheria Toxin/Human B-Cell Activating Factor Fusion Protein Kills Human Acute Lymphoblastic Leukemia BALL-1 Cells: An Experimental Study

Objective： This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor （DT388sBAFF） in Escherichia coli （E. coli） and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells （BALL-1）. Methods： A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction （PCR） and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot, and then purified by Ni2＋-NTA affinity chromatography. The expression level of B cell-activating factor receptor （BAFF-R） on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide （MTT） assay. Results： The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. Conclusion： The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

B超引导下肉毒素注射配合电针治疗痉挛型偏瘫前臂旋前患儿的临床效果

目的分析B超引导下肉毒素注射配合电针治疗痉挛型偏瘫前臂旋前患儿的临床效果。方法选取2018年1月至2021年11月收治的60例痉挛型偏瘫前臂旋前患儿为研究对象,按照随机数字表法将其分为对照组和治疗组,各30例。对照组采用针刺及作业疗法,治疗组采用B超引导下肉毒素注射后,给予电针、作业疗法治疗。比较两组的临床疗效。结果治疗后,两组的Ashworth分级和评分均明显改善,且治疗组优于对照组(P<0.05)。治疗后,两组患儿的精细运动能力测试量表(FMFM)和粗大运动功能测试量表(GMFM)评分均明显提高,且治疗组高于对照组(P<0.05)。治疗后,两组患儿的上肢功能测试(UEFT)评分和前臂旋后角度均明显改善,且治疗组优于对照组(P<0.05)。治疗组的治疗总有效率高于对照组(P<0.05)。结论运用B超引导下肉毒素注射配合电针治疗痉挛型偏瘫前臂旋前患儿可快速准确地降低上肢肌张力,缓解肌肉痉挛,改善上肢运动功能,提高治疗效果。

倪海雯应用芪苓白头翁汤治疗脾虚湿热型原发肠道弥漫大B细胞淋巴瘤临证经验

总结倪海雯治疗原发肠道弥漫大B细胞淋巴瘤的经验。原发肠道弥漫大B细胞淋巴瘤病情凶险,预后差,临床表现缺乏特异性,治疗棘手。倪海雯以周仲瑛癌毒学说及病机十三条理论为指导,从“虚、毒、湿、热”复合病机论治,归纳本病核心病机为“脾虚癌毒,挟杂湿热”,并结合不同治疗阶段采用分期论治、病证结合的中西整合模式,在经典名方白头翁汤基础上结合癌毒病机加以创新,创立验方芪苓白头翁汤以健脾益气,燥湿清肠,标本兼治。临床随访观察发现,其可明显改善患者临床症状,减少复发,提高患者生活质量。

霍乱肠毒素B亚单位在转基因番茄果实中特异性表达及其免疫原性的研究

利用番茄果实特异性启动子-E8启动子,将霍乱肠毒素B亚单位基因(ctb)用根癌农杆菌浸润法转入番茄植株,并使其在果实中特异表达,然后对试验小鼠进行口服免疫,研究转基因番茄果实的免疫原性。结果表明,ctb基因在14株番茄植物基因组中被证实有整合,并在其中2株的番茄果实中有特异性表达,最高表达量分别为455和385 ng·g-1FW,口服免疫后的小鼠血液和肠道粘膜中均检测到抗CTB抗体。表明获得了在番茄果实中特异、高效表达霍乱肠毒素B亚单位基因(ctb)的植物口服疫苗候选植株。

霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达

目的构建霍乱弧菌肠毒素B亚单位(Cholera toxin B subunit,CTB)基因的大肠杆菌表达重组质粒,并观察其在大肠杆菌和双歧杆菌中的表达。方法从pBI121质粒PCR扩增获得CTB基因片断,克隆到大肠杆菌载体pGEX-4T-1上,构建重组质粒,然后转化大肠杆菌DH5α和双歧杆菌。转化菌经IPTG诱导,然后用SDS-PAGE和Western blot方法鉴定表达的重组蛋白。结果构建了重组质粒pGEX-4T-CTB,CTB基因片段分子量约为376bp;在大肠杆菌中表达出35kD的霍乱弧菌B亚单位融合蛋白,经SDS-PAGE分析,相对分子量与文献相符,表达的蛋白约占细菌总蛋白的10%;在双岐杆菌中也能得到正确表达,表达量较大肠杆菌低,占细菌总蛋白约5%。Western blotting结果确认了该条带为CTB基因的产物。结论构建的重组质粒pGEX-4T-CTB能够在大肠杆菌及双歧杆菌中获得表达。

霍乱肠毒素B亚单位在转基因番茄中表达的研究

将霍乱肠毒素 B亚单位 (CT-B)基因及内质网引导序列 (SEKDEL)克隆到质粒 p RTL2和 p BI1 2 1中 ,分别构建植物双元表达载体 p BI-CTB和 p BI-CTBK,CT-B基因由 Ca3 5 S启动子控制表达 .采用叶盘法经根癌农杆菌介导转化番茄 (金丰 1号 ,Jinfeng1 ) ,各表达载体得到一批转基因植株 .经 PCR和 Southern blot分析表明 CT-B基因整合到了番茄基因组中 ;ELISA和 Western blot分析表明 p BI-CTB和 p BI-CTBK的转基因植株能够有效表达 CT-B多肽 ,分别占番茄叶片可溶性蛋白的 0 .0 5 5 %和 0 .0 84% .

大肠杆菌志贺毒素stxB融合基因的构建及其高效表达

利用 PCR方法 ,从肠出血性大肠杆菌 O15 7∶ H7的基因组 DNA中 ,分别扩增出 Stx1B和 Stx2 B亚基的结构基因片段 ,然后再通过 PCR将这 2个片段连接起来 ,并定向克隆到表达质粒 p ET2 8a的 Nco 和 Eco R 位点之间 ,构建了重组表达质粒 p MB1。将重组质粒转化到宿主菌 BL2 1(DE3)中 ,对重组菌株 BL2 1(p MB1)用 IPTG进行诱导表达 ,并对最佳诱导时间进行了探索。 SDS- PAGE电泳检测结果表明 ,重组菌株表达出了 16 30 0的目的蛋白 Stx2 B- L-Stx1B;经薄层扫描分析表明 ,表达量约占菌体总蛋白的 6 8.4 % ,且目的蛋白为包涵体表达 ,表达量约占细菌沉淀的84 .6 %。研究还表明 ,未用 IPTG诱导的 BL2 1(p MB1)菌株也可有少量目的蛋白的基础表达。不同诱导时间的结果表明 ,在 3～ 7h的诱导时间内 。

痢疾志贺氏毒素B亚单位在大肠杆菌中的高效表达

本文从痢疾志贺氏Ⅰ型菌W30864的染色体克隆了编码志贺氏毒素(Stx)的基因,表达Stx的基因位于约4.5kb的EcoR1片段内。一系列的生物学试验表明:我们构建的杂种质粒(pMGC001)能产生Stx,产量为亲代株的16倍,克隆株不仅有细胞毒和肠毒作用,而且还有神经毒性。我们又从质粒pMGC001将志贺氏毒素的B亚单位(Stx-B)的基因克隆至表达载体pJLA503,获得了Stx-B在大肠杆菌中的高效表达,Stx-B已被纯化,其特异的多克隆和单克隆抗体也被制备。Western blot表明它们能与Stx-B进行特异的抗原抗体反应。

猪圆环病毒2型Cap蛋白部分基因序列与大肠杆菌LTB成熟肽基因的融合表达及免疫原性研究

为了获得猪圆环病毒2型(Porcine circovirus type 2,PCV-2)衣壳蛋白(Cap)基因3’端396 bp的片段与大肠杆菌不耐热肠毒素B亚单位(LTB)成熟肽编码区融合基因的高效表达,并检验重组蛋白的免疫保护效果。以含PCV-2全基因组的pMDT PCV-2质粒为模板,用PCR的方法扩增PCV-2 Cap基因,并将其克隆到pET28a(+)中,构建得到pET-Cap质粒,利用EcoRⅠ和HindⅢ双酶切切下Cap基因3’末端396 bp的片段,插入到pET-LTB原核表达质粒LTB基因的3’末端,从而构建pET-LTB△Cap原核表达质粒,将其转化大肠杆菌BL21(DE3)pLysS后,用IPTG诱导重组菌,用SDS-PAGE电泳和Western blot检测诱导物。表达产物经初步纯化后用昆明系小白鼠做免疫保护实验。结果表明:pET-LTB△Cap重组融合表达质粒在大肠杆菌中实现了高效表达,融合蛋白分子量约为29Ku,表达量约占菌体蛋白的36.67%,融合蛋白能被PCV-2阳性血清识别。攻毒后,所有小鼠临床表现正常。用PCR检测有1/8的免疫小鼠感染了PCV-2,而对照组8只小鼠都感染了PCV-21。PCV-2 Cap基因3’端396 bp的片段与LTB基因融合能实现高效表达,表达产物免疫的小白鼠可抵抗PCV-2的攻击此研究为PCV-2基因工程疫苗的研究奠定了基础。