结核分支杆菌Ag85B基因在大肠杆菌中的高效表达

目的 通过结核分支杆菌Ag85B蛋白编码基因在大肠杆菌中稳定表达，以获得大量纯化的Ag85B蛋白。方法 采用DNA重组技术构建结核分支杆菌Ag85B基因的表达载体，双酶切和聚合酶链反应(PCR)鉴定重组子，阳性重组子转化大肠杆菌，并诱导表达外源蛋白。十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)鉴定Ag85B蛋白抗原在大肠杆菌中的表达，对染色的凝胶扫描，以测定目的蛋白表达水平。结果 菌体蛋白经SDSPAGE，含阳性重组质粒的菌体蛋白中出现一条新蛋白带，表达量占菌体总蛋白的33％～38％。Ag85B蛋白抗原在大肠杆菌中表达方式主要是包涵体形式。结论 构建的大肠杆菌重组体能高效表达结核分支杆菌Ag85B蛋白抗原。     

结核分支杆菌Ag85B蛋白在大肠杆菌中的高效表达

目的：通过结核分支杆菌Ag85B基因在大肠杆菌中的表达，获得大量纯化的重组Ag85B(rAg85B)蛋白。方法:应用PCR技术扩增结核分支杆菌H37RvAg85B DNA序列；以质粒pET24b为表达载体，构建Ag85B重组质粒，然后转化大肠杆菌BL21(DE3)；以IPTG诱导转化子，通过SDS—PAGE电泳鉴定rAg85B的表达水平，采用Novagen公司生产的His．Bind蛋白纯化试剂盒纯化rAg85B蛋白。结果:重组质粒PET24b—Ag85B测序表明与报道的序列相同；它在大肠杆菌BL21(DE3)细胞内以包涵体形式存在，表达量占菌体总蛋白的30％左右。纯化后的rAg85B样品经SDS—PAGE和光密度扫描分析表明其纯度约为95％左右，每100ml培养菌可获得10mg左右纯化的重组蛋白。结论:pET24b—Ag85B大肠杆菌工程菌株能高效表达rAg85B蛋白，大量纯化的rAg85B为其进一步的研究与应用奠定了基础。     

人睾丸新基因SPAG4L的原核表达与纯化

目的 原核表达人睾丸生精相关基因SPAG4L,并对重组蛋白进行纯化,为下一步研究SPAG4L的生物学功能奠定基础.方法 运用RT-PCR从人睾丸中扩增编码SPAG4L126-379的基因片段,并将PCR产物克隆至pUCm-T载体.用限制性内切酶EcoRⅠ和HindⅢ消化后,将目的 片段克隆至带有6个组氨酸标签的原核表达载体PQE30中.重组质粒PQE-30-SPAG4L经酶切和测序验证后,转化大肠杆菌JM15,IPTG诱导融合蛋白表达.SPAG4L融合蛋白以westernblot进行鉴定,最后用NI-NTA磁性琼脂糖珠进行纯化.结果 成功构建了重组表达质粒PQE-30-SPAG4L,并能够在大肠杆菌JM15中诱导表达,经Westernblot分析证实,IPTG诱导表达的是SPAG4L融合蛋白.建立小规模的SPAG4L融合蛋白表达、纯化系统,获得了带有6个组氨酸标签的纯化的SPAG4L融合蛋白.结论 成功地对人睾丸生精相关基因SPAG4L进行了体外原核表达,获得了纯化的融合蛋白蛋白,为进一步研究该蛋白在精子发生中的生物功能奠定了基础.     

结核分枝杆菌Ag85B/IL-2融合蛋白的原核表达、纯化、鉴定及活性初步测定

目的:在大肠杆菌中表达结核分枝杆菌Ag85B/IL-2融合蛋白,并对其进行纯化、鉴定和活性初步测定. 方法:将构建的pGEX-Ag85B/IL-2重组菌BL21扩增后接种于LB培养基中,异丙基硫代半乳糖苷(IPTG)诱导重组融合蛋白的表达,筛选最适诱导剂浓度和最适诱导时间;梯度浓度尿素复性包涵体,经GST-Sepharose亲和层析、凝血酶切、阴离子交换层析和反相-高效液相(RP-HPLC)层析纯化Ag85B/IL-2融合蛋白,免疫印迹(Western blot)鉴定,并测定其N-末端氨基酸序列和IL-2比活性. 结果:成功在大肠杆菌中高效表达带有GST的Ag85B/IL-2融合蛋白,占菌体总蛋白的30%,主要以包涵体形式表达,于诱导后6 h表达量达最高峰. 对包涵体复性,纯化获得纯度为98.32%的Ag85B/IL-2融合蛋白,Western blot鉴定阳性,N-末端氨基酸测序与理论预期完全一致,IL-2比活性为2500 u/mg. 结论:高效表达并纯化了Ag85B/IL-2融合蛋白,为进一步研究其在膀胱肿瘤免疫治疗中的作用奠定了基础.     

结核杆菌Ag85B的基因克隆及在大肠杆菌中的表达

目的：通过结核杆菌Ag85B基因的克隆和在大肠杆菌表达的研究,探讨Ag85B蛋白作为候选疫苗用于结核免疫预防和治疗的可行性。方法：用PCR技术,扩增人结核杆菌Edman株Ag85B基因序列,并将扩增的DNA片段插入克隆质粒载体pUC18,用质粒酶谱和DNA充阢分析鉴定重组克隆。克隆的Ag85B基因插入原核表达质粒pBV220,构建重组Ag85B表达质粒,用SDS－PAGE和Western blot鉴定大肠杆菌表达的重组蛋白。结果：成功克隆了结核杆菌Ag85B基因,构建了Ag85B的原核表达载体,获得了高效表达菌株,目的蛋白的表达量达菌总蛋白的25％－30％。结论：获得了高效表达人结核杆菌Ag85B成熟蛋白的工程菌株,为Ag85B保护性抗原位点分析和新型结核疫苗的研制奠定了基础。     

分枝杆菌Ag85B多克隆抗体的研制

目的在原核系统中表达并纯化GST-Ag85B融合蛋白,制备兔抗Ag85B多克隆抗体.方法首先构建编码结核分枝杆菌Ag85B分泌蛋白的重组表达质粒pGEX-Ag85B,将此质粒转入大肠杆菌,诱导表达融合蛋白并加以纯化;然后将纯化的蛋白免疫家兔,提取家兔的抗血清,纯化制备多克隆抗体并进行鉴定.结果成功构建了原核表达质粒pGEX-Ag85B,转化入大肠杆菌BL21中诱导表达并成功纯化,用纯化的GST-Ag85B融合蛋白成功制备了兔抗Ag85多克隆抗体.结论制备的多克隆抗体为进一步的研究奠定了基础.     

人釉原蛋白的原核表达、纯化及鉴定

目的 构建含人釉原蛋白(hAm)成熟肽基因的重组原核表达质粒,在大肠杆菌中表达重组人釉原蛋白,鉴定经纯化获得的融合蛋白.方法 利用限制性内切酶EcoR I和Sal I将hAm基因插入原核表达载体PGEX4T1,对重组表达质粒PGEX4T1-hAm进行双酶切和测序鉴定.选择测序正确的质粒转化E.coli.RosettaTM,用终浓度0.3 mmol/L的IPTG诱导表达带有亲和标记谷胱甘肽-S-转移酶(GST)的重组hAm.经亲和层析纯化获得融合蛋白GST-hAm,并通过SDS-PAGE和Western blotting鉴定原核表达的重组hAm.结果 重组质粒经双酶切和测序鉴定证实插入序列准确无误.在大肠杆菌RosettaTM中经IPTG诱导表达并纯化得到的融合蛋白GST-hAm,经SDS-PAGE鉴定分子量约为46 000,主要以包涵体形式存在,与预测一致;Western blotting证实该融合蛋白能被特异性抗hAm多克隆抗体识别.结论 成功构建含有hAm成熟肽基因的重组原核表达质粒,表达的融合蛋白GST-hAm可通过纯化获得,经鉴定为hAm.大量纯化的hAm成熟肽的获得为开展其功能研究奠定了基础.     

人釉原蛋白的原核表达、纯化及鉴定

目的构建含人釉原蛋白(hAm)成熟肽基因的重组原核表达质粒,在大肠杆菌中表达重组人釉原蛋白,鉴定经纯化获得的融合蛋白。方法利用限制性内切酶EcoRⅠ和SalⅠ将hAm基因插入原核表达载体PGEX4T1,对重组表达质粒PGEX4T1-hAm进行双酶切和测序鉴定。选择测序正确的质粒转化E.coli.RosettaTM,用终浓度0.3 mmol/L的IPTG诱导表达带有亲和标记谷胱甘肽-S-转移酶(GST)的重组hAm。经亲和层析纯化获得融合蛋白GST-hAm,并通过SDS-PAGE和Western blotting鉴定原核表达的重组hAm。结果重组质粒经双酶切和测序鉴定证实插入序列准确无误。在大肠杆菌RosettaTM中经IPTG诱导表达并纯化得到的融合蛋白GST-hAm,经SDS-PAGE鉴定分子量约为46000,主要以包涵体形式存在,与预测一致;Western blotting证实该融合蛋白能被特异性抗hAm多克隆抗体识别。结论成功构建含有hAm成熟肽基因的重组原核表达质粒,表达的融合蛋白GST-hAm可通过纯化获得,经鉴定为hAm。大量纯化的hAm成熟肽的获得为开展其功能研究奠定了基础。     

HIV-1 SF2株env基因C1与C3区的嵌合表达

①目的 构建HIV-1SF2株env基因C1与C3区嵌合片段的克隆并在大肠杆菌中表达。②方法 用DNAstar软件辅助设计引物，以含有HIV-1SF2株前病毒基因组的9B1R6质粒为模板，PCR法分别扩增出env基因C1和C3段；两个片段经KpnⅠ酶切、T4连接酶连接和扩增后，再将经EcoRⅠ、SalⅠ双酶切的嵌合片段插入表达载体pGEX-4T-2中，构建重组表达质粒pGEX-4T-2／ClC3；重组质粒经酶切、测序鉴定后，以TSS法转入大肠杆菌DH5α，IPTG诱导表达融合蛋白和SDS—PAGE电泳分析表达蛋白图谱。③结果 经酶切和测序分析，插入片段大小、方向和读码框架正确，无碱基错配，表明成功构建pGEX-4T-2／ClC3克隆；重组克隆在大肠杆菌DH5α中经IPTG诱导，高效表达出相对分子质量为4．3万的融合蛋白。④结论 重组质粒pGEX-4T-2／C1C3的构建和表达成功，为进一步研究该表达蛋白的活性和HIV疫苗的研制奠定了基础。     

结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合基因的克隆表达及生物信息学分析

目的构建结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合表达载体,并进行抗原的生物信息学分析,为候选疫苗筛选奠定基础。方法从结核分枝杆菌H37Rv菌株中分别扩增出Ag85B基因,TB10.4基因,Ag85B-TB10.4;将Ag85B克隆入pET-28a(+)表达载体中,将TB10.4和Ag85B-TB10.4分别克隆入pET-SUMO表达载体中。上述重组质粒转化入大肠埃希杆菌BL21菌中(DE3),经IPTG诱导表达,并对其进行生物信息学分析。结果 (1)成功将结核分枝杆菌Ag85B和TB10.4,Ag85B-TB10.4基因分别克隆入pET28a(+)和pET-SUMO表达载体中,经IPTG诱导蛋白表达后,对经超声裂解的菌液上清进行SDSPAGE电泳,表明获得了与预期蛋白大小一致的表达产物,重组融合蛋白pET-SUMO-Ag85B-TB10.4蛋白分子质量约为54kDa。(2)生物信息学分析显示Ag85B-TB10.4融合蛋白具有多个潜在抗原位点。结论成功构建了重组质粒pET-28a-Ag85B,pET-SUMO-TB10.4和pET-SUMO-Ag85B-TB10.4,并获得了Ag85B,TB10.4和Ag85B-TB10.4的可溶性原核表达产物;生物信息学分析在Ag85B与TB10.4蛋白加入一段蛋白linker后蛋白的亲水性增强,抗原决定簇面积增大,为后续的功能实验研究奠定了基础。     

肱骨远端全骺分离

肱骨远端全骺分离在5岁以下儿童中是较常见的肘部损伤,且极易误诊为肱骨髁上骨折或肘关节脱位。本组25例肱骨远端全骺分离均为向内侧移位,年龄11个月至14岁,平均5.6岁,属Salter-HarrisⅠ型或Ⅱ型。对本症骨骺损伤的解剖学和组织学,诸骨(和骨化中心)之间的对应关系以及骨骺损伤的诊治进行了简短的讨论。     

食指背侧皮瓣的临床应用

通过对右手虎口挛缩以及左拇指皮肤剥脱损伤各一例应用直指背侧皮瓣修复的成功经验,提出该手术的适应症和优点,并重点介绍其应用解剖学和手术方法。     

Investigation of hypoglycemic,hypolipidemic and antioxidant activities of aqueous extract of Terminalia paniculata bark in diabetic rats

Objective

To investigate the hypoglycemic, hypolipidemic and antioxidant activities of aqueous extract of Terminalia paniculata bark (AETPB) in streptozotocin (STZ)-induced diabetic rats.

Methods

Acute toxicity was studied in rats after the oral administration of AETPB to determine the dose to assess hypoglycemic activity. In rats, diabetes was induced by injection of STZ (60 mg/kg, i.p.) and diabetes was confirmed 72 h after induction, and then allowed for 14 days to stabilize blood glucose level. In diabetic rats, AETPB was orally given for 28 days and its effect on blood glucose and body weight was determined on a weekly basis. At the end of the experimental day, fasting blood sample was collected to estimate the haemoglobin (Hb), glycosylated haemoglobin (HbA1c), serum creatinine, urea, serum glutamate-pyruvate transaminase (SGPT), serum glutamate-oxaloacetate transaminase (SGOT) and insulin levels. The liver and kidney were collected to determine antioxidants levels in diabetic rats.

Results

Oral administration of AETPB did not exhibit toxicity and death at a dose of 2 000 mg/kg. AETPB treated diabetic rats significantly (P<0.001, P<0.01 and P<0.05) reduced elevated blood glucose, HbA1c, creatinine, urea, SGPT and SGOT levels when compared with diabetic control rats. The body weight, Hb, insulin and total protein levels were significantly (P<0.001, P<0.01 and P<0.05) increased in diabetic rats treated with AETPB compared to diabetic control rats. In diabetic rats, AETPB treatment significantly reversed abnormal status of antioxidants and lipid profile levels towards near normal levels compared to diabetic control rats.

Conclusions

Present study results confirm that AETPB possesses significant hypoglycemic, hypolipidemic and antioxidant activities in diabetic condition.     

Association of smoking,alcohol and NSAIDs use with expression of cag A and cag T genes of Helicobacter pylori in salivary samples of asymptomatic subjects

Objective

To determine the association of smoking, alcohol and nonsteroidal anti-inflammatory drugs (NSAIDs) use with presence and virulence of Helicobacter pylori (H. pylori) infection in a representative sample of a random adult population of asymptomatic subjects.

Methods

Non virulent 16S rRNA and virulent cag A and T genes from salivary samples of 854 asymptomatic subjects were determined using polymerase chain reaction. The presence and absence of virulent and non virulent infection was statistically compared with consumption of smoking, alcohol and NSAIDs.

Results

The prevalence of infection in male and female subjects was found to be 69.25% and 66.90%, respectively. The prevalence of infection in the population of asymptomatic subjects with respect to consumption of alcohol was as follows: current (31.22%), former (52.20%) and never (43.58%). The prevalence of infection in the population of asymptomatic subjects with respect to smoking of cigarettes was as follows: current (88.80%), former (57.14%) and never (33.33%). The prevalence of infection in the subject population consuming NSAIDs and not consuming NSAIDs frequently was found to be 82.75% and 21.16%, respectively. Virulence in male and female subjects was found to be 60.00% and 50.00%, respectively. The presence of virulent infection in the population of asymptomatic subjects with respect to consumption of alcohol was as follows: current (28.57%), former (40.15%) and never (50.00%). The prevalence of virulent infection in the population of asymptomatic subjects with respect to smoking of cigarettes was as follows: current (79.32%), former (75.00%) and never (50.00%). The prevalence of virulent infection in the subject population consuming NSAIDs and not consuming NSAIDs frequently was found to be 88.23% and 66.66%, respectively.

Conclusions

It can be concluded that smoking and NSAIDs consumption are aggravating factors for virulence of H. pylori and alcohol can inhibit H. pylori infection in asymptomatic subjects.     

大肠多发原发性癌:危险因素、诊治和预防(附8例报告)

综述分析1954～84年国内外文献,探讨了大肠多发原发性癌的诊断标准、发病情况、临床表现、发病危险因素、外科处理以及预防和诊断检测技术等问题,强调了大肠腺瘤,遗传基础、免疫缺陷、慢性溃疡性结肠炎等在发病上的作用,及高危险组患者应按方案进行常规检测监护之重要性。述介了大肠多发原发性癌系一种原发性大肠癌多中心发生的形式,在诊治上前者与后者有一致性,亦各有其独特性。报告宁夏医学院附属医院肿瘤科1977～84年收治的8例住院大肠多发原发性癌病例的临床资料,对诊治经过进行了初步总结。     

具有抗细菌黏附和杀菌双重功能的(HA/CHI-Van)5涂层的制备

目的：研究开发一种涂覆(HA/CHI-Van)5多层膜结构的骨科内植入物以用于预防骨内植入物感染。方法：将透明质酸（HA）和壳聚糖-万古霉素（CHI-Van）溶液进行层层自组装,制备得到(HA/CHI-Van)5多层膜结构;通过zeta电位检测组装过程,通过活细菌染色实验评价多层膜的抗细菌黏附效果,通过越狱实验和FE-SEM实验检测体外抗菌效果。SD大鼠随机分为空白组、(HA/CHI-Van)5组和假手术组,并以X线、Micro-CT、骨组织切片等方法检测该材料的体内动物实验效果。结果：每次组装不同的单层,zeta电位交替变化,证明多层膜组装成功。活细菌染色实验显示多层膜结构中的CHI具有一定的抗细菌黏附作用。越狱实验和FE-SEM实验证明(HA/CHI-Van)5多层膜结构在体外具有良好的抗菌作用。在体外实验中,(HA/CHI-Van)5组的X线评分为0.7,而空白组的评分为12.3,(HA/CHI-Van)5组在骨密度、骨小梁数量、骨体积分数、骨组织切片方面均优于空白组（均P＜0.05）,且非常接近假手术组（均P＞0.05）。结论：通过在骨科内植入物表面涂覆(HA/CHI-Van)5多层膜结构能够获得一定的抗细菌黏附功能和良好的抗菌作用,可以预防骨内植入物感染。     

Efficacy and side effects of praziquantel in the treatment of Schistosomiasis mansoni in schoolchildren in Shesha Kekele Elementary School,Wondo Genet,Southern Ethiopia

Objective

To evaluate the efficacy and side effects of praziquantel (PZQ) in the treatment of schistosomiasis in Ethiopia.

Methods

In a cross-sectional study, stool specimens were collected from randomly selected 299 school children in Shesha Kekele Elementary School, Wondo Genet, Southern Ethiopia, in April 2010. Stool specimens were examined using a single Kato-Katz thick smear for Schistosoma mansoni (S. mansoni) ova. Children who were found positive for S. mansoni were treated with a single oral dose of PZQ at 40 mg/kg bw and interviewed for treatment-related symptoms 24 hours after drug administration. Four weeks post-treatment, stool specimens were collected from the same children and examined following the same procedure as in the pre-treatment. Drug efficacy was determined based on cure and egg reduction rates.

Results

Pre-treatment prevalence of S. mansoni infection was 74.9% with geometric mean egg count of 268. The evaluated generic PZQ produced an overall cure rate of 73.6% (P<0.000 1, OR: 8.33, CI: 5.3–13.1) and egg reduction rate of 68.2% (P=0.03, F=0.64). The cure rate showed significant association with age (χ²=11, P=0.004), the highest rate being observed in the 15–22 age group. 83% of S. mansoni infected children showed various treatment-related symptoms, the most frequent being headache, nausea, and abdominal pain. These symptoms were associated with age (P<0.001) and pre-treatment intensity of infection (P<0.05).

Conclusions

The present observations revealed relatively lower cure and egg reduction rates of the PZQ evaluated as compared to previous reports for other PZQ brands in Ethiopia. Hence, in depth studies are recommended to clarify whether the present relatively lower cure rate is the actual cure rate of the praziquantel evaluated, treatment failure, or reduced susceptibility of the parasite. Treatment-related side effects observed were transient and tolerable.     

NGR修饰唑来膦酸长循环脂质体制备及其表征研究

目的:制备具有肿瘤血管靶向的NGR修饰唑来膦酸长循环脂质体,并对其进行表征研究。方法：采用薄膜分散法制备NGR修饰唑来膦酸长循环脂质体,,并对其进行表征研究,并测定包封率。结果：制备得到的NGR-PEG-ZOL-LPs平均粒径为(271.43±9.23)nm,Zeta电位为-(36.20±0.20)mv,多分散系数(PDI)为(0.15±0.03),理化性质相对稳定;NGR-PEG-ZOL-LPs包封率为8.41%;电镜下显示NGR-PEG-ZOL-LPs呈类圆形,大小分布均匀,无明显聚集。结论：采用薄膜分散法制备的NGR修饰唑来膦酸长循环脂质体具有较高的稳定性,从而为体内药效学研究奠定基础。     

Control of Tetranychus urticae Koch by extracts of three essential oils of chamomile,marjoram and Eucalyptus

Objective

To evaluate the acaricidal activity of extracts of three essential oils of chamomile, marjoram and Eucalyptus against Tetranychus urticae (T. urticae) Koch.

Methods

Extracts of three essential oils of chamomile, marjoram and Eucalyptus with different concentrations (0.5%, 1.0%, 2.0%, 3.0% and 4.0%) were used to control T. urticae Koch.

Results

The results showed that chamomile (Chamomilla recutita) represented the most potent efficient acaricidal agent against Tetranychus followed by marjoram (Marjorana hortensis) and Eucalyptus. The LC₅₀ values of chamomile, marjoram and Eucalyptus for adults were 0.65, 1.84 and 2.18, respectively and for eggs 1.17, 6.26 and 7.33, respectively. Activities of enzymes including glutathione-S-transferase, esterase (α-esterase and β-esterase) and alkaline phosphatase in susceptible mites were determined and activities of enzymes involved in the resistance of acaricides were proved. Protease enzyme was significantly decreased at LC₅₀ of both chamomile and marjoram compared with positive control. Gas chromatography-mass spectrometer (GC-MS) proved that the major compositions of Chamomilla recutita are α-bisabolol oxide A (35.251%), and trans-β-farersene (7.758%), while the main components of Marjorana hortensis are terpinene-4-ol (23.860%), p-cymene (23.404%) and sabinene (10.904%).

Conclusions

It can be concluded that extracts of three essential oils of chamomile, marjoram and Eucalyptus possess acaricidal activity against T. urticae.