柯萨奇病毒B3(CVB3)通过激活NADK2促进小鼠巨噬细胞NLRP3的活化

目的研究在柯萨奇病毒B3(CVB3)刺激下,小鼠巨噬细胞中含pyrin结构域NOD样受体家族蛋白3(NLRP3)表达的变化及机制。方法CVB3刺激RAW264.7细胞、小鼠原代(骨髓来源或腹腔)巨噬细胞以及感染NLRP3短发夹RNA(shRNA-NLRP3)慢病毒的RAW264.7细胞,实时荧光定量PCR检测NLRP3和白细胞介素1β(IL-1β)表达水平;ELISA检测细胞上清中IL-1β的含量;Western blot法检测NLRP3蛋白的变化,免疫共沉淀(Co-IP)法检测NLRP3相互作用蛋白的表达。结果CVB3刺激RAW264.7细胞、骨髓来源或腹腔巨噬细胞后,NLRP3和IL-1β表达明显升高;下调NLRP3后,IL-1β分泌明显减少;NLRP3抗体Co-IP所得复合物银染,组间差异蛋白经质谱分析鉴定为烟酰胺腺嘌呤二核苷酸激酶2(NADK2),证明CVB3诱导NADK2与NLRP3相互作用。结论CVB3通过激活NADK2促进巨噬细胞NLRP3活化,增加炎症因子IL-1β表达和释放。

Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress

Objective Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3(CVB3) infection induced autophagy through endoplasmic reticulum(ER) stress. Methods In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction(RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase(PERK) inhibitor, inositol-requiring protein-1(IRE1) inhibitor, or activating transcription factor-6(ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3(LC3). Results CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein(GFP)-LC3 punctuation and induced the conversion from LC3-Ⅰ to phosphatidylethanolamine-conjugated LC3-1(LC3-Ⅱ). CVB3 infection still decreased the expression of mammalian target of rapamycin(mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-Ⅱ to LC3-Ⅰ in CVB3-infected HeLa cells. Conclusion CVB3 infection induced autophagy through ER stress in HeL a cells, and PERK, IRE1, and ATF6 a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

Coxsackievirus B3-induced apoptosis and Caspase-3

Cell death can be classified into two categories: apoptosis and necrosis. Apoptotic pathway can beeither caspase-dependent or caspase-independent. Caspase-independent cytopathic effect (CPE) has beendescribed. In order to evaluate the pattern of HeLa cell death induced by Coxsackievirus B3 (CVB3)and whether apoptosis involves caspase activation, we co-cultivated HeLa cells with CVB3 and detectedthe cytopathic changes, the alteration of mRNA and protein expression of caspase-3 gene plus caspase-3activity, as well as analyzing DNA fragmentation before and after caspase-3 activity inhibition. Accordingto the results, we propose that CVB3 may induce apoptosis and necrosis in HeLa cells, the latter appearingmuch earlier. Caspase-3 is activated at the levels of both transcription and translation, and procaspase-3 isproteolytically cleaved, thus leading to the continuous increasing of both caspase-3 precursor protein and itssubunit. However, besides CPE, apoptosis induced by CVB3 is not a direct consequence of the activationof caspase-3, or caspase-3 is not the only effector molecule in apoptotic cell death, for caspase-3 inhibitorcan not decrease DNA fragmentation. Some other biochemical mechanisms may participate in the process,whose role weakens the effect of inhibiting caspase-3 activity.

In vitro anti-coxsackievirus B_3 effect of ethyl acetate extract of Tian-hua-fen

AIM: To investigation the anti-coxsackievirus B3 (CVB3m)effect of the ethyl acetate extract of Tian-hua-fen on HeLacells infected with CVB3m.METHODS: HeLa cells were infected with CVB3m and thecytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A600 was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A600.RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration.Consistent results were got through these two methods.We also investigated the mechanism of its anti-CVB3m effectand the results indicated that the extract represented an inhibitory effect through all the processes of CVB3m attachment, entry, biosynthesis and assemble in cells.CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significantprotectiveeffect on HeLa cells infected with CVB3m in a dose-dependent manner and this effect exists through the process of CVB3mattachment, entry, biosynthesis and assemble in cells,suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.

柴胡 黄芩 炙甘草对小鼠CVB3心肌炎治疗作用的研究

目的 探讨单剂柴胡、黄芩、炙甘草对柯萨奇病毒B3(CVB3)感染的病毒性心肌炎小鼠的治疗作用。方法 用CVB3感染Balb/C小鼠造成病毒性心肌炎模型 ,分别用生理盐水、柴胡、黄芩、炙甘草灌胃治疗 ,观察病毒感染后不同时间点的心肌组织病理变化。结果 柴胡组小鼠在感染后第 5 ,7,10天心肌病理积分为 1.33±0 .82 ,1.6 0± 0 .6 3和 1.87± 0 .74 ,明显低于对照组 (2 .2 2± 1.37,3.2 7± 0 .96 ,2 .6 0± 1.0 6 ) (P <0 .0 5或 0 .0 1)。炙甘草组小鼠在感染后第 5 ,7天心肌病理积分为 1.2 6± 0 .88和 1.80± 0 .6 8,明显低于相应对照组 (P <0 .0 5或0 .0 1)。黄芩组小鼠在感染第 14 ,2 1天后心肌病理积分 (1.5 3± 0 .74 ,0 .80± 0 .5 6 )与对照组 (2 .33± 0 .98,1.4 0±0 .83)比较差异有显著性 (P <0 .0 5 )。结论 单剂柴胡、黄芩。

EFFECT OF COXSACKIEVIRUS B3 ON ION CHANNEL CURRENTS IN RAT VENTRICULAR MYOCYTES

To investigate the effects of coxsackievirus B 3(CVB 3) on ion channel currents in rat ventricular myocytes. Methods.Rat hearts were isolated with collagenase to acquire single ventricular myocytes, L type voltage dependent calcium channel(VDCC)current (I Ca ),Na + current (I Na ), outward potassium current (I out ), inwardly rectifying potassium current(I KI ) were recorded using whole cell patch clamp techniques. ResultsCVB 3 infection increased I Ca and I out , while decreased I KI ; but it had no obvious effect on I Na . Conclusion.The effects of CVB 3 on I Ca 、 I out 、 I KI may be one of the mechanisms of myocytes damage and the occurrence of abnormal electroactivities induced by CVB 3 infection.

抗柯萨奇B病毒性心肌炎胶囊复方新制剂抗CVB_3作用

目的通过体内外实验观察抗柯萨奇B病毒性心肌炎胶囊K-CoXB-JN复方新制剂对柯萨奇B3(CVB3)病毒感染的心肌细胞和病毒性心肌炎(VMC)小鼠的保护作用。方法体外实验用1000 TCID50的CVB3感染心肌细胞后,分别加入不同浓度的K-CoXB-JN复方新制剂和利巴韦林,并设定正常对照组和模型对照组,当细胞病变达到75%后,测定细胞上清液中肌酸激酶(CK)、天冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)活性;体内实验用CVB3感染Balb/c小鼠造成病毒性心肌炎模型,分别予K-CoXB-JN复方新制剂高、中、低剂量,利巴韦林和纯水连续灌胃14 d,记录小鼠死亡数,观察药物对小鼠的死亡保护情况。结果 K-CoXB-JN复方新制剂可降低CVB3感染的心肌细胞培养上清液中CK、AST、LDH含量,降低病毒性心肌炎小鼠的死亡率,给药组和模型对照组相比较P<0.05。结论 K-CoXB-JN复方新制剂对CVB3感染的心肌细胞和病毒性心肌炎小鼠具有保护作用。

免疫途径和佐剂对CVB3VP1蛋白免疫效果的影响

目的:观察不同免疫途径和佐剂类型对柯萨奇病毒B组3型(Coxsackievirus group B type 3,CVB3)衣壳蛋白VP1免疫效果的影响。方法:将原核表达质粒pET-his/VP1转入E.coli BL21(DE3)pLysS中,用异丙基-1-硫代-β呋喃半乳糖苷(IPTG)诱导CVB3VP1蛋白的表达并进行纯化。首先采用不同的免疫途径(皮下,腹腔,肌肉)用VP1蛋白免疫小鼠,每组12只。然后另取小鼠分为PBS组和不同佐剂组(氢氧化铝、弗氏佐剂、Montanide ISA720),每组18只,采用肌肉注射途径免疫。每次每只小鼠注射50μg,共免疫3次,间隔3周。用ELISA和微量中和试验检测血清特异性IgG抗体和中和抗体。用CCK-8法检测淋巴细胞增殖活性和CTL杀伤活性。用致死量的CVB3攻击后,检测血中病毒的滴度并观察小鼠的存活状况。结果:在大肠杆菌中成功表达CVB3VP1蛋白。三种免疫途径比较,肌肉注射组血清中和抗体和特异性IgG抗体的水平明显高于其他组(P<0.01)。采用肌肉注射免疫时,弗氏佐剂组Montanide ISA 720佐剂组的体液免疫和细胞免疫应答的水平明显高于氢氧化铝组(P<0.05);但血中病毒的滴度低于氢氧化铝组(P<0.05)。弗氏佐剂组小鼠的生存率好于氢氧化铝组(P<0.05)。结论:采用肌肉注射途径,并联合弗氏佐剂或Montanide ISA 720佐剂可以使CVB3VP1免疫获得较好的免疫效果。

抗柯萨奇B病毒性心肌炎胶囊复方新制剂对CVB_3感染小鼠的治疗作用

目的观察抗柯萨奇B病毒性心肌炎胶囊(K-CoxB-JN复方新制剂)对CVB3感染小鼠的治疗作用。方法用CVB3感染Balb/c小鼠造成病毒性心肌炎模型,分别予K-CoxBJN复方新制剂高、中、低剂量,利巴韦林和纯水灌胃十四天。观察小鼠一般状态、记录死亡数,分别在第7、14天每组随机抽取小鼠标号,称体质量,取血测天冬氨酸转氨酶(AST)、肌酸激酶(CK)含量活性;之后取心,脾,胸腺称重,计算脏器指数;取心脏放入10%甲醛中固定,HE染色观察心肌病理变化,记录病理积分。结果 K-CoxB-JN复方新制剂能降低病毒性心肌炎小鼠的死亡率和血清中CK、AST含量,给药组和模型对照组相比较P<0.05,并能降低心脏指数和心脏病理积分,但对脾、胸腺指数影响不明显。结论 K-CoxB-JN复方新制剂对CVB3感染的小鼠有治疗作用。

IL-15基因真核表达质粒的构建及其对CVB3 VP1基因疫苗的免疫增强作用

目的探讨人白细胞介素 15 (humaninterleukin 15 ,hIL 15 )基因佐剂对柯萨奇B3病毒 (CoxsackievirusB3 ,CVB3 )VP1基因疫苗的免疫增强效果。方法从人胚肺二倍体细胞提取hIL 15的mRNA ,用逆转录 聚合酶链反应 (reversetranscriptase polymerasechainreaction ,RT PCR)扩增出cDNA ,构建成真核表达克隆 pcDNA3 IL 15。将pcDNA3 VP1、pcDNA3 VP1+ pcDNA3 IL 15、pcDNA3 IL 15、pcDNA3和盐水分别肌内注射Balb/c小鼠 ,每次免疫后 6天取血清用微量中和试验测CVB3中和抗体 ,3次免疫后用 80 0TCID5 0CVB3感染小鼠 ,观察小鼠的生存率。结果 pcDNA3 IL 15重组质粒的目的基因与hIL 15基因序列相同 ;pcDNA3 VP1和pcDNA3 VP1+ pcDNA3 IL 15能诱导小鼠产生中和抗体 ,抗体滴度随免疫次数增加而提高 ;pcDNA3 VP1+ pcDNA3 IL 15组抗体滴度和生存率均高于 pcDNA3 VP1组。结论本研究成功构建了hIL 15重组质粒 ,该质粒对真核表达重组质粒 pcDNA3

Evaluation of Antibodies Induced by the Injection of Single Capsid Protein or Purified Virus Particle of Coxsackievirus B3 in Mice

Four capsid proteins (VP1, VP2, VP3, and VP4) of coxsackievirus B3 (CVB3) were expressed as recombinant proteins in an Escherichia coli expression system and used as antigens for subunit vaccines against CVB3 in ICR mice. Antigens were expressed as thioredoxin-histidine (TrxHis)-tagged protein and purified before immunization. Although all VPs other than VP4 induced anti-CVB3 specific antibodies in mice (detected by ELISA and western blotting), they did not neutralize the infectious CVB3 in a virus neutralization assay. Meanwhile, 2 virus strains were purified from CVB3 virus stock on the basis of their plaque size on HeLa cells. ICR mice were infected with the 2 purified virus strains (S-strain and L-strain) and unpurified virus stock (wild type) to analyze the difference in antibody responses against infections of purified and unpurified virus strains. The reactivity of antisera against each virus strain was tested by ELISA, and the results showed that the inoculation of purified virus strain induced a strong antibody response against the inoculated strain. As a result, the antibody response against wild-type and other virus strains was suppressed. These results suggest using unpurified virus stock as an antigen is advantageous for inducing a broad antibody response in inoculated animals.

ROLE OF COXSACKIEVIRUS AND ADENOVIRUS RECEPTOR (CAR)IN CARDIOTOXICITY INFECTED BY COXSACKIEVIRUS B3

Objective To explore the role of coxsackievirus and adenovirus receptor（CAR） in cardiotoxicity infected by coxsackieviras B3. Methods A toxic cellular model was established in vitro by adding myocarditic coxsackievirus B3 （CVB3m） into the culture of neonatal mouse cardiomyocytes. 48 h later, the cardiomyocytes were divided into control, CVB3m, and CAR antibody ＋ CVB3m groups. CVB3m-mediated myocytopathic effect of above three groups was observed after further culturing for 48h. At the same time, the cardiomyocytes＇ viability of above three groups was assessed by MTT assay. Results The degree of cytopathic effect（CPE） of CAR antibody ＋ CVB3m group was significantly lower than CVB3m group （ P 〈 0. 01 ） and there was a significant increase in cell viability in CAR antibody ＋ CVB3m group compared with CVB3m group（ P 〈 0. 01 ）. No significant difference was found between CAR antibody ＋ CVB3m group and control group. Conclusion CAR antibody possesses a protective effect on CVB3m infected cardiomyoctyes, which indicates that CAR may play an important role in mediating cardiotoxicity infected by CVB3m.

PROTECTIVE ROLE OF SOPHOCARPINE IN COXSACKIEVIRUS B3 INFECTION IN CULTURED RAT CARDIOMYOCYTES

Objective To observe the anti-CVB3 （ Coxsackievirus B3 ） effect of sophocarpine （SC） extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultured to establish the viral myocarditis model The cells were divided into four groups： infected group （ infected by CVB3 ） , SC treated group （ added SC 100 μg/mL after viral infection ）, SC control group （ added SC 100 μg/mL only）, and normal control group. The cytopathic effect （CPE） and the beating frequency of the myocardial cells were observed and the LDH levels in the supernatant were measured at day 2,3, and 5. The cultured myocytes were added different concentrations of SC （ 12. 5 -400 μg/mL ） after infection with CVB3, the CPE was observed and the concentrations of LDH were measured and compared at day 2, 3, and 5. Results In the SC treated group （ 100 μg/mL ） , the cytopathic effect was lighter and the LDH level was lower than the infected group. SC in a concentration of 12. 5 - 300 μg/mL could relieve the CPE and lower the LDH level, while in a higher concentration （400 μ/m ） , it exacerbated the CPE caused by the virus, and the LDH levels were higher than the infected cells. Conclusion SC in certain concentration could protect the cultured rat cardiomyocytes from CVB3 infection.

柴胡提取物对CVB3m感染小鼠心肌细胞凋亡相关基因表达的影响

目的探讨柴胡提取物调节心肌细胞凋亡的作用机制.方法以柯萨奇病毒嗜心肌株(CVB3m)腹腔注射诱导BALB/c小鼠病毒性心肌炎模型,将实验小鼠分为正常对照组、模型组、柴胡提取物组,柴胡提取物组每天予0.2ml/10g的浓度为2mg/ml的柴胡提取物灌胃,正常对照组、模型组予以等量生理盐水灌胃,于造模后第10天处死,采用TUNEL法检测心肌细胞凋亡,采用免疫组化、RT-PCR法检测各组小鼠心肌中Fas、Bcl-2、Caspase3表达.结果正常组小鼠心肌未检测到凋亡的心肌细胞,Fas、Bcl-2、Caspase3均有不同程度表达,模型组可见大量心肌细胞凋亡,且Fas、Bcl-2、Caspase3表达均强于对照组(P＜0.01),柴胡提取物组心肌细胞凋亡明显减少,与模型组比较差异显著(P＜0.01),Fas、Caspase3表达明显降低,Bcl-2表达轻度升高.结论柴胡提取物可以抑制病毒性心肌炎时心肌细胞凋亡,明显下调Fas及Caspase3表达,轻微上调Bcl-2表达.

苦参碱对CVB_3感染CMVECs的保护作用

目的:研究苦参碱对CVB_3感染CMVECs的保护作用。方法:采用倒置显微镜观察CMVECs形态,CCK8检测细胞活性,选择苦参碱最佳干预时间点和稀释浓度。在最佳条件下通过免疫荧光法、western blot法检测内皮细胞标志物CD31的表达。结果:苦参碱最佳干预时间点和浓度是:72 h,0.012 5 mg/L。与空白对照组比较,CVB_3组CMVECs形态固缩、变异,内皮细胞标志物CD31表达减少;与CVB_3组比较,CVB_3+苦参碱组CMVECs形态趋于正常,CD31表达增强。结论:苦参碱对CVB_3感染CMVECs具有保护作用,增加内皮细胞功能可能是其作用机制。

Coxsackievirus B3 HFMD animal models in Syrian hamster and rhesus monkey

Coxsackievirus B3(CVB3)is the pathogen causing hand,foot and mouth disease(HFMD),which manifests across a spectrum of clinical severity from mild to severe.However,CVB3-infected mouse models mainly demonstrate viral myocarditis and pancreatitis,failing to replicate human HFMD symptoms.Although several enteroviruses have been evaluated in Syrian hamsters and rhesus monkeys,there is no comprehensive data on CVB3.In this study,we have first tested the susceptibility of Syrian hamsters to CVB3 infection via different routes.The results showed that Syrian hamsters were successfully infected with CVB3 by intraperitoneal injection or nasal drip,leading to nasopharyngeal colonization,acute severe pathological injury,and typical HFMD symptoms.Notably,the nasal drip group exhibited a longer viral excretion cycle and more severe pathological damage.In the subsequent study,rhesus monkeys infected with CVB3 through nasal drips also presented signs of HFMD symptoms,viral excretion,serum antibody conversion,viral nucleic acids and antigens,and the specific organ damages,particularly in the heart.Surprisingly,there were no significant differences in myocardial enzyme levels,and the clinical symptoms resembled those often associated with common,mild infections.In summary,the study successfully developed severe Syrian hamsters and mild rhesus monkey models for CVB3-induced HFMD.These models could serve as a basis for understanding the disease pathogenesis,conducting pre-trial prevention and evaluation,and implementing post-exposure intervention.

Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis

Viral myocarditis(VMC)is one of the most common acquired heart diseases in children and teenagers.However,its pathogenesis is still unclear,and effective treatments are lacking.This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes(CMCs)induced by coxsackievirus B3(CVB3).CVB3 was utilized for inducing the VMC mouse model and cellular model.Cardiac echocardiography,left ventricular ejection fraction(LVEF),and left ventricular fractional shortening(LVFS)were implemented to assess the cardiac function.In CVB3-induced VMC mice,cardiac insufficiency was observed,as well as the altered levels of ferroptosis-related indicators(glutathione) peroxidase 4(GPX4),glutathione(GSH),and malondialdehyde(MDA).However,exosomes derived from human umbilical cord mesenchymal stem cells(hucMSCs-exo)could restore the changes caused by CVB3 stimulation.Let-7a-5p was enriched in hucMSCs-exo,and the inhibitory ffect of hucMSCs-exoa-ie-pmimo on CVB3-induced ferroptosis was higher than that of hucMSCs-exommie N(NC:negative control).Mothers against decapentaplegic homolog 2(SMAD2)increased in the VMC group,while the expression of zinc-finger protein 36(ZFP36)decreased.Let-7a-5p was confirmed to interact with SMAD2 messenger RNA(mRNA),and the SMAD2 protein interacted directly with the ZFP36 protein.Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators.Meanwhile,the levels of GPX4,solute carrier family 7,member 11(SLC7A11),and GSH were lower in the SMAD2 overexpression plasmid(oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group,while those of MDA,reactive oxygen species(ROS),and Fe^(2+)increased.In conclusion,these data showed that ferroptosis could be regulated by mediating SMAD2 expression.Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36,which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

大蒜多糖体外抗柯萨奇病毒B_3作用

目的 研究大蒜多糖体外抗柯萨奇病毒B3 (CoxsackievirusgroupBtype3, CVB3 )作用。方法 观察大蒜多糖A,B,C的细胞毒性、对CVB3 直接灭活作用、抗CVB3 吸附作用及对CVB3 生物合成抑制作用。结果 大蒜多糖A,B,C对Hep 2细胞的半数中毒浓度(TC50 )分别为 1000, 800, 4000μg/mL;均无直接灭活CVB3 及抗CVB3 吸附作用;除A外,B和C均可剂量依赖性抑制CVB3 生物合成.。结论 大蒜多糖B和C在体外通过抑制CVB3 生物合成而发挥抗CVB3 的作用。

黄芪对柯萨奇B_3病毒核糖核酸作用的研究及其机理探讨

以体外转录合成、同位素３５Ｓ标记的肠道病毒特异负股ＲＮＡ为探针，对急性柯萨奇Ｂ３病毒（ＣＶＢ３）心肌炎小鼠心肌中的ＣＶＢ３－ＲＮＡ进行ＲＮＡ－ＲＮＡ原位杂交检测，利用图像分析仪对阳性杂交信号进行图像处理与定量分析；观察黄芪对ＣＶＢ３－ＲＮＡ含量的影响；同时对黄芪的抗病毒机理及其与β－干扰素的关系进行了初步探讨。结果发现在病毒感染的心肌组织中，黄芪组心肌坏死面积明显小于生理盐水对照组。黄芪对ＣＶＢ３－ＲＮＡ的复制有良好的抑制作用，其机制与β－干扰素（β－ＩＦＮ）无关。

柯萨奇B3病毒VP1基因在原核中的表达及其临床意义

目的:研究柯萨奇B3病毒(CVB3)VP1基因在大肠杆菌中的表达及其临床意义。方法:将柯萨奇B3病毒中国分离株VP1基因克隆入PQE-30载体,转导入大肠杆菌(Ml5)中,使CVB3的VP1基因在大肠杆菌中得到稳定的表达,采用NAT亲和方法纯化,间接ELISA方法做免疫学活性鉴定。结果:表达的VP1产物与抗柯萨奇B3病毒的兔抗CVB3(多克隆抗体)血清产生较强的免疫反应,与天然的CVB3蛋白抗原(病毒组织培养抗原)的抗原性近似。应用无关兔抗体对照呈阴性反应。应用表达的VP1产物作为抗原,对临床急性病毒性心肌炎患者血清进行了特异性IgM酶联免疫吸附试验检测,其结果与组织培养的CVB3制备的细胞蛋白抗原对上述患者血清的特异性IgM检测结果基本一致。结论:采用基因工程方法制备出的CVB3-VP1蛋白抗原产量高,纯化后免疫反应原性基本没有变化。试用表达CVB3的VP1基因工程抗原代替目前实验用有感染危险的病毒培养抗原的方法,快速、特异地检出CVB3的感染,为急性心肌炎的早期诊断和及时的临床治疗提供可靠的实验室诊断指标。