HPV16型L1蛋白优势表位原核表达及其血清学验证

目的利用原核系统对人乳头瘤病毒(HPV)16型L1蛋白优势抗原表位进行重组表达与纯化,并检测血清学反应。方法对HPV16型L1蛋白优势抗原表位进行全基因合成,将核酸片段克隆至pET-DsbC原核表达载体,重组质粒转化大肠埃希菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用亲和层析对表达产物进行纯化,采用免疫印迹法、酶联免疫吸附试验(ELISA)验证重组蛋白的免疫学活性。结果成功构建了pET-DsbC-HPV16 L1表达载体,并在大肠埃希菌Rosetta(DE3)中实现了稳定的可溶性表达。重组DsbC-HPV16 L1蛋白经亲和层析进行纯化,相对分子质量为45 000,纯度为95%,重组蛋白纯化得率为22%。免疫印迹法结果显示,重组DsbC-HPV16 L1蛋白可与HPV16型阳性血清产生特异的抗原-抗体反应,与重组DsbC蛋白或健康人血清无交叉反应,具有良好的特异性。ELISA结果显示,重组DsbC-HPV16 L1蛋白与HPV16型阳性血清有较强的免疫反应性,A450 nm均值为0.56,高于健康人血清和磷酸盐缓冲液(PBS)阴性对照(A450nm均值分别为0.24和0.21)。结论采用原核表达系统实现了HPV16型L1蛋白优势抗原表位的可溶性高表达,获得的重组蛋白具有良好的免疫原性和免疫学活性。     

白念珠菌烯醇化酶重组蛋白质的制备及鉴定

目的 制备具有高免疫原性的白念珠菌重组烯醇化酶蛋白质。 方法 以白念珠菌C1标准株基因组DNA作为模板,用PCR法扩增烯醇化酶的全长DNA序列,以pET28a(+)为载体,构建重组表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导重组融合蛋白质表达。用抗his标签的单克隆抗体和白念珠菌抗体阳性的病人血清进行western blot鉴定,亲和层析柱纯化重组蛋白质。 结果 获得了含白念珠菌烯醇化酶全长基因的重组表达载体和相应工程菌株,经IPTG诱导后能高效表达重组融合蛋白质。经已含抗体的病人血清进行western blot鉴定,表明诱导的重组蛋白质有良好的免疫原性。 结论 成功克隆了白念珠菌烯醇化酶全长基因并在大肠埃希菌中获得高效表达;重组蛋白质具有良好的免疫原性。     

白念珠菌烯醇化酶N端肽段ENO1 319P的原核表达和鉴定

摘要：目的:制备白念珠菌烯醇化酶N端基因重组肽段ENO1-319P并检测其抗原性。 方法:选择与人烯醇化酶蛋白α-ENO同源性低的白念珠菌烯醇化酶肽段ENO1-319P,将其编码DNA序列克隆到表达载体pET28a(+),并在大肠埃希菌BL21(DE3)中用IPTG诱导带有His标签的重组肽段的表达。用SDS-PAGE电泳分析,TALON金属亲和层析柱纯化重组蛋白,其抗原性用侵袭性白念珠菌感染（ICAI）患者血清进行western blot评估。 结果:成功表达并纯化了目的肽段,该蛋白可与ICAI患者血清发生特异性反应。 结论:获得了白念珠菌烯醇化酶N端肽段ENO1-319P的基因表达工程菌株,为将此重组肽段用于制备特异性更高的抗体打下基础。     

人巨细胞病毒pp150-gp52蛋白原核可溶性表达与IgM捕获ELISA方法建立和应用

目的原核系统内表达并纯化巨细胞病毒pp150-gp52融合蛋白优势抗原表位,建立IgM捕获ELISA方法并应用。方法通过重叠PCR技术扩增获得巨细胞病毒pp150-gp52优势片段核酸序列,在原核系统内可溶性表达DsbC-pp150-gp52融合蛋白并纯化,用Western blot和ELISA检测融合蛋白的特异性和应用价值。结果纯化获得的融合蛋白DsbC-pp150-gp52经酶标记建立IgM捕获ELISA方法,检测60份临床阳性血清和60份健康人血清。其中以酶标记DsbC-pp150-gp52蛋白建立的捕获ELISA法阳性检出率96.7% ,阴性检出率100%,初步验证DsbC-pp150-gp52融合肽具有非常好的抗原特异性。结论融合蛋白DsbC-pp150-gp52在大肠埃希菌中以可溶性表达形式存在,获得的高纯度重组融合蛋白具有抗原性和特异性强的特点,采用IgM捕获ELISA的实验方法,可开发检测试剂盒用于风疹病毒的早期检测。     

梅毒螺旋体嵌合抗原的克隆表达及其临床应用

目的 在大肠埃希菌中表达梅毒螺旋体Tp15—17嵌合抗原,用于临床检测梅毒感染。方法 利用PCR法从Tp全基因组中分别扩增目的基因Tp15和Tp17,用T4DNA连接酶连接后,克隆进表达载体pGEX-6P-1中,构建重组质粒pGEX-6P-1＋Tp15-17,在大肠埃希菌中表达重组Tp嵌合蛋白,重组蛋白经亲和层析柱纯化后包被微孔板,初步建立间接ELISA法检测血清中的抗Tp抗体。结果 重组嵌合抗原获得了高效表达,免疫印迹试验显示重组抗原具有很强的抗原性。用重组嵌合抗原建立间接ELISA法,对梅毒螺旋体抗体阳性参比血清及梅毒患者血清的检测符合率100％。结论 重组梅毒螺旋体嵌合抗原Tpl5—17能够作为梅毒螺旋体诊断性ELISA抗原。     

梅毒螺旋体Tp0319重组蛋白的表达、纯化及其在临床诊断中的初步应用

目的克隆、表达及纯化梅毒螺旋体(Tp)外膜蛋白Tp 0319,并建立间接ELISA方法,探讨其在梅毒血清学诊断中的价值。方法构建重组质粒PQE32/Tp 0319,IPTG诱导表达,SDS-PAGE和western blot鉴定表达结果;Ni-NTA亲和层析法纯化重组蛋白,间接ELISA检测梅毒患者血清中特异性IgG抗体。结果成功构建了PQE32/Tp 0319重组质粒;经SDS-PAGE检测,诱导产物显示有一条相对分子质量约为30 000的特异蛋白质条带,western blot分析证明其只与人抗Tp IgG发生特异性反应;将Tp 0139用于间接ELISA检测抗Tp抗体阴、阳性参比血清,特异性和敏感性均为100%;检测梅毒患者和健康献血者血清各150份,结果与TPPA法符合率为95.3%。结论制备的Tp 0319重组蛋白有较好的免疫反应性,可望用于临床梅毒血清学诊断。     

结核分枝杆菌Rv3618蛋白在大肠埃希菌中的表达、纯化及其血清学诊断

目的 大肠埃希菌表达系统获重组结核分枝杆菌Rv3618蛋白,并评价其用于肺结核病血清学诊断的价值.方法 大肠埃希菌表达重组Rv3618蛋白,离子和疏水层析纯化获得高纯度的重组Rv3618蛋白,ELISA检测71例肺结核患者血清重组Rv3618蛋白和重组38000蛋白抗体IgG.结果 Rv3618蛋白在大肠埃希菌中表达,从大肠埃希菌纯化获得的重组Rv3618蛋白和重组38000蛋白的ELISA诊断临界值分别为A490 nm=0.51和A 490 nm=0.45.71例肺结核患者血清,重组Rv3618蛋白抗体阳性率39.4%与重组38000蛋白抗体阳性率(52.1%)比较差异无统计学意义(P＞0.05),而与两种重组蛋白联合检测的抗体阳性率(59.2%)比较差异有统计学意义(P＜0.05).结论 重组Rv3618蛋白可与重组38000蛋白联合用于肺结核病患者血清诊断以提高敏感性,因此,重组Rv3618蛋白可作为结核病血清学诊断的抗原之一.     

重组人胱抑素C及其多克隆抗体的制备

目的探讨重组人胱抑素C（Cys C）及其多克隆抗体的制备。方法根据大肠埃希菌编码蛋白的特性设计Cys C编码基因序列,人工合成目的基因,并将其在大肠埃希菌中表达,目的蛋白经亲和层析纯化后免疫新西兰大白兔;采用Western blot和酶联免疫吸附测定（ELISA）法检测兔血清重组人Cys C及其多克隆抗体。结果制备的重组人Cys C纯度达90%以上,抗重组人Cys C抗体具有很好的结合效价和特异性。结论制备的重组人Cys C及其抗体可用于进一步的实验研究和临床应用。     

免疫印迹法测定Tp47蛋白与梅毒患者血清的免疫反应性

目的克隆Tp47基因、构建原核表达载体、表达并纯化Tp47蛋白,通过免疫印迹法(Western-Blot)检测其免疫反应活性。方法聚合酶链反应扩增Tp47基因,将Tp47克隆至pGEX-6P-1载体,构建重组表达质粒pGEX-6P1-Tp47,经测序鉴定正确后,转化大肠埃希菌BL21(DE3),经诱导表达后,用亲和层析方法纯化重组蛋白,鉴定正确后采用Western-Blot检测重组蛋白与梅毒阴性、阳性血清的免疫反应性。结果成功构建pGEX-6P1-Tp47原核表达质粒,表达、纯化后获得了相对分子质量约为71×103的重组蛋白;Western-Blot检测显示其能与梅毒阳性患者血清发生特异性反应,而与梅毒阴性患者血清无交叉反应性。结论成功克隆、表达、纯化Tp47重组蛋白,其与临床各期梅毒患者血清具有较好的免疫反应性,为Tp47重组蛋白用于梅毒早期诊断提供了理论依据。     

酶切与PCR联用合成梅毒抗原TpN17全长DNA及其表达活化和ELISA检测

目的在大肠埃希菌中重组表达梅毒密螺旋体TpN17抗原，用其建立梅毒诊断血清学方法。方法酶切与PCR联用。合成全长TpN17基因，亚克隆后克隆到pET-HIS质粒并转化到大肠杆菌BL21株中表达。亲和层析纯化。纯化rTpN17包被微孔板，ELISA方法检测临床8份梅毒阳性血清和4份阴性血清。结果获得了重组表达并纯化的TpN17，ELISA检测结果表明重组蛋白能被梅毒患者阳性血清所识别．特异性100％．灵敏度100％。结论证明了该重组蛋白具有良好的免疫反应性，可用于梅毒感染的血清学检测。     

The Hereditary Syndrome of Thrombocytopathia, Bleeding Tendency, Extreme Miosis, Muscular Fatigue, Asplenia, Headache, etc

SYNOPSIS
A new, autosomal dominantly inherited syndrome with a bleeding disorder was described in 1985 by Stormorken and his co-workers.1,2 In this multifaceted syndrome, there were the following integral components: thrombocytopathia, extreme miosis with Argyll Robertson-like traits, muscular fatigue, a tendency to spasms, asplenia, ichthyosis, dyslexia, etc.
Headache with migraine traits was also present in the family in all 4 generations in which this syndrome had been observed. Nasal and conjunctival bleeding were part of the headache picture in some of the individuals exhibiting the hemorrhagic syndrome. While the attack-related bleeding disturbances only involved family members who also suffered from the hemorrhagic syndrome, the headache per se may seem to behave differently: The affected son's headache seems to have developed into a headache with tension headache traits, whereas the other, unaffected, son's headache has common migraine traits. The familial headache which in earlier generations clearly had migraine traits, therefore, may be inherited independently from the hemorrhagic disorder. In other words, a migraine or migraine-like headache is most probably not an obligatory integral part of this syndrome. The thrombocytopathia in this disorder comprises abnormal serotonin storage, uptake, and release (Stormorken and co-workers, to be published).
The admittedly somewhat farfetched possibility also exists that the headache, although being similar to migraine, differs essentially from it and may be an expression of the serotonin aberration.     

Effect of 5-fluorouracil, mitoxantrone, methotrexate, and vincristine on the antibacterial activity of ceftriaxone, ceftazidime, cefotiam, piperacillin, and netilmicin

Using the checkerboard agar dilution technique, antibacterial activity and in vitro interactions of 4 antineoplastic agents and 5 antimicrobial drugs were examined against 56 strains of 7 bacterial species. 5-fluorouracil was found to inhibit all strains of Staphylococcus aureus and of Staphylococcus epidermidis at a concentration of 0.8 micrograms/ml or less. 84% of all gram-negative strains were inhibited synergistically when 5-fluorouracil was combined with beta-lactam antibiotics. Methotrexate and cefotiam were antagonistic in 42% of all combinations, especially when tested against Escherichia coli and Klebsiella pneumoniae.     

Susceptibilities of non-Pseudomonas aeruginosa gram-negative nonfermentative rods to ciprofloxacin, ofloxacin, levofloxacin, D-ofloxacin, sparfloxacin, ceftazidime, piperacillin, piperacillin-tazobactam, trimethoprim-sulfamethoxazole, and imipenem.

Agar dilution MICs of 10 agents against 410 non-Pseudomonas aeruginosa gram-negative nonfermentative rods were determined. MICs at which 50 and 90% of the isolates were inhibited, respectively, were as follows (in micrograms per milliliter): sparfloxacin, 0.5 and 8.0; levofloxacin, 1.0 and 8.0; ciprofloxacin, 2.0 and 32.0; ofloxacin, 2.0 and 32.0; D-ofloxacin, 32.0 and > 64.0; ceftazidime, 8.0 and 64.0; piperacillin with or without tazobactam, 16.0 and > 64.0; trimethoprim-sulfamethoxazole, 0.5 and > 64.0; imipenem, 2.0 and > 64.0. With the exception of those for Stenotrophomonas maltophilia, Burkholderia cepacia, and Alcaligenes faecalis-A. odorans, agar dilution MICs for all strains tested were within 1 dilution of inhibitory (bacteriostatic) levels as determined by time-kill methodology.     

品管圈在提高儿科住院患儿服药到口率中的应用

目的：探讨品管圈在儿科病房服药到口护理工作改进中的管理作用。方法：通过品管圈活动步骤,运用质量管理工具对儿科住院患儿服药不能到口的问题进行改进,并将改进前后状况进行比较讨论。结果：住院患儿服药到口率从42.5%提升到99.19%(P＜0.01)。结论：品管圈方法的应用提高了住院患儿服药到口率,提高了医护人员的团队精神,增强了护士的责任心,提升了护士的个人素质和自信心。     

Comparative ototoxicity of ribostamycin, dactimicin, dibekacin, kanamycin, amikacin, tobramycin, gentamicin, sisomicin and netilmicin in the inner ear of guinea pigs

Nine aminoglycoside antibiotics, ribostamycin (RSM), dactimicin (DAC), dibekacin (DKB), kanamycin (KM), amikacin (AMK), netilmicin (NTL), tobramycin (TOB), gentamicin (GM) and sisomicin (SISO) were administered intramuscularly to guinea pigs for 4 weeks, and ototoxicity and drug concentration in the inner ear fluid were determined. RSM and DAC showed the weakest ototoxicity against the cochlea and vestibular organs. AMK and KM were more toxic to cochlea than vestibular organs. DKB, TOM, GM and SISO were equally toxic to vestibular organs and cochlea. NTL was more toxic to vestibular organs than cochlea. As judged from the pinna reflex response and hair cell damage in the cochlea, the order of auditory toxicity was the following: SISO greater than GM greater than TOB greater than AMK greater than DKB greater than KM greater than NTL, DAC RSM, whereas the vestibular toxicity was in the following order: SISO greater than GM greater than DKB greater than TOB greater than NTL greater than AMK greater than KM greater than DAC, RSM. RSM, causing the weakest ototoxicity, showed a low drug concentration in the inner ear fluid, while GM, causing severe ototoxicity, showed the highest drug level under the same conditions.