Mutated recombinant human glucagon-like peptide-1 induces differentiation of PC12 cells

Glucagon-like peptide-1 （GLP-1） and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited the application of GLP-1 in the clinic. We generated a mutated form of human GLP-1 （mGLP-1） using site-directed mutagenesis and gene recombination techniques, and found that these modifications significantly prolonged the biological half-life of GLP-1 compared with native GLP-1 （nGLP-1）. This study investigated the role of mGLP-1 on inducing PC12 cell differentiation, mGLP-1 induced PC12 cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43 and neuronal class III I^-tubulin, and significantly increased cyclic adenosine monophosphate level. No significant difference was found between mGLP-1 and nGLP-I. The results indicate that mGLP-1 activates the GLP-1 receptor, induces PC12 cell differentiation, and has neurotrophic effects.

Anti-tumor effect of CTLs activated by dendritic cells pulsed with K-ras mutant peptide and whole tumor antigen on pancreatic cancer

Objective： We studied the role of specific cytotoxic T lymphocytes （CTLs） activated by dendritic cells （DCs） presenting cationic nanoparticles with the K-ras （12-Val） mutant peptide and whole tumor antigen in the killing of different pancreatic cancer cell lines in vitro and in vitro. Methods： Peripheral blood DCs were induced by rhGM-CSF and IL-4 and cultured. DCs were sensitized by whole antigen of a pancreatic cancer cell line （PANC-1） with expression of K-ras mutant, K-ras mutant peptide （K-ras＋peptide） and cationic nanoparticles with K-ras mutant peptide （K-ras＋peptide-CNP）, respectively. Cell surface markers were measured by flow cytometry. Lymphocyte proliferation was detected by the 3H-TdR test, and ELISAwas performed to detect IFN-y secretion. 125I-UdR was used to measure the killing effect of CTLs. We also evaluated the antitumor activity of CTLs in vivo in a tumor-bearing nude mouse model prepared with the PANC-1 （K-ras＋） and SW1990 （K-ras-） cell lines. Results： Compared with K-ras＋peptide, low concentration K-ras＋pepUde-CNP can be effectively presented by DCs （P 〈 0.05）. CTLs induced by DCs pulsed with whole tumor antigen had significant greater killing effect （P 〈 0.05） on PANC-1 and SW1990 pancreatic cancer cells compared with K-ras＋peptide and K-ras＋peptide-CNP-induced CTLs. CTLs induced by DCs pulsed with K-ras＋peptide and K-ras＋peptide-CNP had a specific killing effect （P 〈 0.05） for PANC-1 and no effect （P 〉 0.05） on SW1990 cell lines （P 〉 0.05）. Conclusion： Cationic nanoparticles with K-ras （12-Val） mutant peptide can be effectively presented by DCs at a low concentration in a short time. CTLs induced by K-ras＋peptide-CNP had specific killing activity for the pancreatic cancer cell line with the K-ras （12-Val） mutant and could significantly inhibit tumor growth and increase the survival time of tumor-bearing nude mice.

Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients

AIM:To identify and assess mutations in the K-ras and BRAF genes in a cohort of Chinese patients with colorectal cancer (CRC) for their association with various clinicopathological parameters and prognosis.METHODS:Genomic DNA was isolated from frozen tissues.Pyrosequencing analysis was conducted to detect mutations in the K-ras (codons 12,13,and 61) and BRAF genes (codon 600).Statistical analysis was carried out using SPSS-15.0 software.RESULTS:Among the 118 colorectal cancer patients,we detected 41 (34.7%) mutations in the K-ras gene.Mutation frequencies at codon 12 and codon 13 were 23.7% (28/118) and 10.2% (12/118),respectively.Only one patient harbored a point mutation at codon 61 (0.8%,1/118).Gender was the only factor that showed an obvious relationship with K-ras gene mutation (female 44.7% vs male 28.2%,P=0.037).Other clinicopathological features,such as age,location of the tumor,tumor differentiation,Tumor,Node and Metastases classification,and the Union for International Cancer Control staging,showed no positive relationship with K-ras gene mutations.No significant correlation was observed between the presence of K-ras mutations (codons 12,13,and 61) and the survival of the patients.BRAF mutations were rare,and only two patients (1.7%) harbored a detectable mutation at codon 600.CONCLUSION:K-ras gene mutation is a common event in our 118 Chinese CRC patients,with an obvious relationship with gender.However,it seems not to be an independent prognostic factor in CRC patients.The BRAF gene is rarely mutated in Chinese CRC patients.

Hypermethylation of CpG island in O^6-methylguanine-DNA methyltransferase gene was associated with K-rasG to A mutation in colorectal tumor

AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas,62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutantallele-specific amplification was used to detect K-rasG to A point mutation in codon 12.RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation,respectively (P = 0.027, P＜0.001). Thirteen of the 19 colorectal tumors with K-rasG to A point mutation in codon 12 had methylated MGMT(P = 0.011). The frequencies of K-rasG to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.

Highly sensitive ECL-PCR method for detection of K-ras point mutation

A highly sensitive electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for K-ras point mutation detection is developed. Briefly, K-ras oncogene was amplified by a Ru(bpy)3(2+) (TBR)-labeled forward and a biotin-labeled reverse primer, and followed by digestion with MvaI restriction enzyme, which only cut the wild-type amplicon containing its cutting site. The digested product was then adsorbed to the streptavidin-coated microbead through the biotin label and detected by ECL assay. The experiment results showed that the different genotypes can be clearly discriminated by ECL-PCR method. It is useful in point mutation detection, due to its sensitivity, safety, and simplicity.

Study of mutations of p53, APC and K-ras genes in 47 cases of intestinalmetaplasia of gastric mucosa

Objective:To study the role of the mutations of p53, APC and K-ras genes in 47 cases of 3 types of intestinal metaplasia (IM) of gastric mucosa. Methods:In 47 cases of IM, exons 5- 8 of p53 and exons 15 of APC were examined with PCR-SSCP and codon 12 of K-ras with PCR-RFLP to detect the existence of any mutations of these structures. Results:Muta- tions of p53, APC and K-ras were found in 29.8% (14/47),6.4% (3/47) and 6.4% (3/47) respectively in our series of patients who consisted of 33 with types I and II and 14 with type III of IM. The mutation rate of p53 was far higher in patients with type III IM (57.1%,8/14) than in those with types I and II IM(18.2%,6/33)(P <0.05). Though the mutation rate of APC and K-ras was also higher in the patients with type III IM than in those with types I and II IM, it was of no statistical significance (P >0.05). In one case of type III IM, mutation of both p53 and K-ras was found. Conclusion: The molecular changes of 3 types of IM are different. The mutation of p53 may be closely related to carcinogenesis in cases of type III IM and it serve as a sign for the early diagnosis of gastric carcinoma.

Detection of K-ras Gene Point Mutation＇s Style in Human Pancreatic Cancer Cell Line PANC-1 by PCR-SSP

Objective： To detect the style of K-ras gene point mutation in human pancreatic cancer cell line PANC-1 and decide the bp sequence of Ras target position interfered by RNA. Methods： Three kinds of special sequence primers （SSP） for polymerase chain reaction （PCR） with regard to the mutation styles （OAT, COT and GOT） at codon 12 of K-ras were used to study the human pancreatic cancer cell line PANC-1. The amplification products were studied with polyacrylamine gel electrophoresis to detect the style of point mutation. Results： The style of K-ras gene point mutation at codon 12 was OAT in human pancreatic cancer cell line. Conclusion： PCR-SSP is rapid, convenient and high specific. The results provide a basis for further gene therapy by RNA interference for pancreatic cancer.

树突细胞负载K-ras突变多肽诱导抗胰腺癌免疫反应

背景与目的:由于大多数肿瘤抗原尚未确定,所以肿瘤的免疫治疗还处于临床探索阶段。k-ras原癌基因在胰腺癌中有特异性的表达,突变位点恒定,不失为一个良好的免疫靶位。本研究探讨以k-ras基因为靶点体外诱导胰腺癌被动免疫治疗的可行性,为临床进行胰腺癌个体化治疗提供实验依据。方法:利用RT-PCR法扩增k-ras基因,通过流式细胞术检测k-ras癌基因突变类型。合成含突变位点的多肽,负载外周血单核细胞来源的树突细胞(dendritic cell,DC),检测其表达率,对T细胞进行激发活化,M TT法检测肿瘤特异性T细胞对胰腺癌细胞Patu8988的杀伤作用。结果:Patu8988胰腺癌细胞株12位密码子点突变为GGT→GTT,12位氨基酸突变为缬氨酸。合成突变多肽能有效地在树突细胞表达突变位点,激发的肿瘤特异性T细胞(cytotoxic T lym phocytes,CTLs)能有效地杀伤相应的肿瘤细胞。结论:突变多肽能有效地诱导免疫细胞杀伤含k-ras突变基因的胰腺癌细胞,从而为胰腺癌的免疫治疗提供实验依据。

不同方法检测K-Ras基因突变与转移性结直肠癌疗效及预后的关系

目的探讨直接测序法和肽核酸钳制PCR(PNA-PCR)法检测K-Ras基因突变状态与转移性结直肠癌患者疗效及预后的相关性。方法收集110例转移性结直肠癌患者的石蜡包埋肿瘤组织,采用直接测序法和PNA-PCR法分别检测肿瘤组织的K-Ras基因第2外显子第12、13密码子的突变状态,并分析其与患者预后的关系。结果直接测序法检测到43例K-Ras基因突变,PNA-PCR法除了检测出这些突变之外,还在直接测序法检测的野生型中发现了10例突变。对K-Ras突变状态与患者的预后分析发现,直接测序法检测的K-Ras野生型及突变型患者的中位生存时间(OS)分别为20.5个月和15.6个月(P=0.067)。PNA-PCR法检测的野生型和突变型患者的中位OS分别为21.3个月和15.8个月(P=0.014)。两种方法检测的野生型与突变型的有效率和无病进展时间(PFS)差异均无统计学意义。按照这两种方法的检测结果分为3组,高突变组、低突变组和野生型组,仅高突变组与野生型组的OS差异有统计学意义(15.6个月vs.21.3个月,P=0.040)。Cox多因素分析显示,ECOG评分(HR=2.70,95%CI:1.39～5.25,P=0.003)和K-Ras丰度(HR=1.52,95%CI:1.52～2.19,P=0.026)与患者的预后相关。结论 K-Ras突变不是以伊立替康或奥沙利铂为主方案的疗效预测因子。PNA-PCR法检测的K-Ras突变状态与患者的预后有关。建议用PNA-PCR法确定野生型患者,而突变型患者则用直接测序法来确定。

肽核酸钳制-PCR/K-ras突变检测方法在结直肠癌组织中的诊断应用

目的确定本实验室建立的肽核酸钳制(PNA)-PCR/K-ras突变检测方法的阳性判断标准,并评价其对结直肠癌组织的诊断价值。方法将含K-ras基因12密码子突变的质粒和野生质粒以不同比例(突变/总体:0,1/3 200,1/1600,1/800,1/400,1/200,1/100)混合作为标准样品,独立配制6个批次并分别进行PNA-PCR/K-ras突变检测,收集Kras突变CT值及K-ras总体CT值,计算ΔCT值(突变CT值-总体CT值),采用ROC曲线分析突变CT值和ΔCT值诊断K-ras突变的最适Cut-off值,联合两者最适Cut-off值,设定该方法的最终阳性判断标准。分别采用该方法和直接测序法对35例结直肠癌组织及对应癌旁组织进行K-ras突变检测并比较分析。结果突变模板浓度为1/800及以上的标准样品突变CT值和ΔCT值与阴性标准品之间差异存在统计学意义(P<0.05);突变CT值和ΔCT值的最适Cut-off值分别为41.7和15.4。最终阳性判断标准为突变CT值≤41.7或ΔCT值≤15.4,对应的ROC曲线下面积为0.955(P=0.001),以此判断标准,各标准样品(0,1/3 200,1/1 600,1/800,1/400,1/200,1/100)的阳性检测率分别为0%、66.7%、83.3%、100%、100%、100%、100%,检测下限为1/800。在结直肠癌及癌旁组织标本中,该方法阳性检测率为45.7%(32/70),与直接测序法(18.6%,13/70)比较差异具有统计学意义(P=0.000)。结论 PNA-PCR/K-ras突变检测方法具有较高的检测灵敏度,在结直肠癌组织样本中具有较直接测序法更高的阳性检出率。

抗MCV抗体、抗CCP抗体对类风湿关节炎的诊断价值及其与疾病活动度的相关性

目的探讨抗突变型瓜氨酸波形蛋白(MCV)抗体、抗环瓜氨酸肽(CCP)抗体对类风湿关节炎(RA)的诊断价值,并分析其与RA疾病活动度的相关性。方法选取74例RA患者(RA组)、95例结缔组织病患者(非RA组)和60例健康体检者(健康对照组)作为研究对象。基于红细胞沉降率的28个关节疾病活动度评分(DAS28-ESR)将RA组患者分为高活动期组(n=22)、中活动期组(n=18)、低活动期组(n=14)和缓解期组(n=20)。比较RA组、非RA组、健康对照组之间的血清抗MCV抗体、血清抗CCP抗体、血清类风湿因子(RF)和ESR阳性率,以及RA不同活动期组之间的血清抗MCV抗体水平、血清抗CCP抗体水平、血清RF水平和ESR。通过绘制受试者工作特征曲线分析血清抗MCV抗体水平、血清抗CCP抗体水平、血清RF水平和ESR单独及联合诊断RA的价值。分析RA组患者血清抗MCV抗体水平、血清抗CCP抗体水平、血清RF水平和ESR与DAS28-ESR的相关性。结果(1)RA组的血清抗MCV抗体、血清抗CCP抗体、血清RF和ESR阳性率高于非RA组和健康对照组(P<0.05);非RA组的血清抗CCP抗体、血清RF和ESR阳性率高于健康对照组(P<0.05),但两组的血清抗MCV抗体阳性率差异无统计学意义(P>0.05)。(2)缓解期组、低活动期组、中活动期组、高活动期组的血清抗MCV抗体水平、血清抗CCP抗体水平和ESR依次升高(P<0.05);高活动期组血清RF水平高于其他3组(P<0.05),其他3组之间的血清RF水平差异无统计学意义(P>0.05)。(3)血清抗MCV抗体水平、血清抗CCP抗体水平单独诊断RA的曲线下面积(AUC)均>0.85,且均大于血清RF水平和ESR的AUC(P<0.05);血清抗MCV抗体水平与血清抗CCP抗体水平诊断RA的敏感度、特异度、AUC差异无统计学意义(P>0.05);血清抗MCV抗体水平、血清抗CCP抗体水平、血清RF水平和ESR联合诊断RA的AUC达0.935,大于任意单一指标诊断RA的AUC(P<0.05)。(4)RA患者的血清抗MCV抗体水平、血清抗CCP抗体水平和ESR与DAS28-ESR均呈正相关(P<0.05)。结论相比于传统指标血清RF和ESR,血清抗MCV抗体和血清抗CCP抗体对RA均有较高的诊断价值;将二者联合血清RF和ESR用于诊断RA,可明显提高诊断效能。血清抗MCV抗体水平、血清抗CCP抗体水平均与RA疾病活动度呈正相关,可作为评估患者疾病活动度的辅助指标。

Unique GGT→GTT mutation at K-ras codon 12 in six human pancreatic cancer cell lines from Chinese patients

Objective To investigate the K-ras mutation pattern in six pancreatic cancer cell lines from Chinese patients. Methods All six cell lines were analyzed for mutations in exon 1 of the K-ras gene by polymerase chain reaction (PCR) and direct sequencing.Results All 6 pancreatic cancer cell lines had GGT→GTT mutations at K-ras codon 12 but no mutations at codon 13.Conclusion The unique GGT→GTT mutation at codon 12 plays a potential role in the carcinogenesis of pancreatic cancers in Chinese.

Evaluation of Intraductal Ultrasonography, Endoscopic Brush Cytology and K-ras, P53 Gene Mutation in the Early Diagnosis of Malignant Bile Duct Stricture

Background： In qualitative diagnosis of bile duct stenosis, single diagnostic measure is difficult to make a correct diagnosis, to combine several diagnostic techniques may be helpful to make an accurate diagnosis. The aim of this study was to evaluate the value of intraductal ultrasonography （IDUS）, endoscopic brush cytology and K-ras, P53 gene mutation in the early diagnosis of malignant biliary stricture. Methods： From February 2012 to February 2013, 84 patients with suspected malignant biliary stricture were performed I DUS firstly, then endoscopic brush cytology and finally K-ras, P53 gene mutation detection, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of all above ways were evaluated and compared. Results： Of 84 patients, 52 cases were ultimately diagnosed malignant biliary stenosis; of which, 9 cases had no recurrence or metastasis to other organs after radical operation during the follow-up period. IDUS combined with brush cytology and K-ras ＋ P53 gene mutation detection had obvious advantage in the sensitivity, accuracy and negative predictive value than any other joint detection and single detection （the advantage was more significant compared with IDUS ＋ brush cytology or any single detection P 〈 0.01）. There were obvious statistical significance in the sensitivity and accuracy between IDUS ＋ brush cytology ＋ P53 or IDUS ＋ brush cytology ＋ K-ras and IDUS ＋ brush cytology or IDUS （P 〈 0.05）. There was no statistical significance in the sensitivity, specificity, positive predictive value, negative predictive value and accuracy between IDUS ＋ brush cytology ＋ P53 and IDUS ＋ brush cytology ＋ K-ras （P 〉 0.05）. Conclusions： IDUS combined with brush cytology and K-ras, P53 gene mutation detection is better than the separate detection and contribute to the early diagnosis of malignant biliary stricture. Its more widespread use is recommended.

Performance of K-ras mutation analysis plus endoscopic ultrasound- guided fine-needle aspiration for differentiating diagnosis of pancreatic solid mass： a meta-analysis

Background Difficulties persist in differentiating pancreatic ductal adenocarcinomas （PDAC） from pancreatic inflammatory masses （PIM）. Auxiliary diagnostic techniques which enhance the endoscopic ultrasound-guided fine-needle aspiration （EUS-FNA） diagnostic yield have been attempted, for example, K-ras mutation analysis. We aimed to evaluate the accuracy of K-ras mutation analysis combined with EUS-FNA for the differential diagnosis of PDAC and PIM by pooling data of existing trials. Methods We systematically searched the Medline, PubMed, Web of Science, Embase, and Cochrane Central Trials databases for relevant published studies. Meta-analysis was performed. Pooling was conducted in fixed-effect model or random-effect model. Results In total eight studies, with 696 cases of PDAC and 138 cases of PIM, met our inclusion criteria. The pooled sensitivity, specificity, positive likely ratio and negative likely ratio of K-ras mutation analysis combined with cytopathology for diagnosis of PDAC versus PIM were 90%, 95%, 13.45, and 0.13, respectively. Especially, among total 123 patients whose EUS-FNA results were inconclusive or negative, fifty-nine had K-ras mutations and were finally diagnosed with PDAC （48%, 59/123）. Publication bias was not present. Conclusions Combining K-ras mutation analysis with routine cytology moderately improves the ability of EUS-FNA to differentially diagnose between PDAC and PIM, especially for patients with suspected PDAC yet inconclusive EUS-FNA findings, and may prove to be a valuable supplemental method to EUS-FNA.

抗真菌多肽——捷安肽素高产菌的选育

从新疆棉株上分离得到一株细菌ZK ,经培养物性状和生理生化鉴定 ,确定该菌为芽孢杆菌属 (Bacillussp.)菌 ,其代谢产物为一种抗病原真菌的肽类物质———捷安肽素 .以此株菌作为出发菌株 ,进行紫外线、微波和亚硝基胍诱变 ,诱变处理后获得高产突变株Mv2 8,在摇瓶试验中 ,该变异株产捷安肽素活性比出发菌株提高 31.6 % .图 5表 5参

Lactoferricin B基因与几种变异克隆的构建及其在酿酒酵母中的表达与鉴定

目的构建牛乳铁多肽(Lactoferricin B)的真核表达质粒pYES2/Lactoferricin B,实现其在酿酒酵母S.cerevisiae中的表达,并初步检测其不同变异的体外抗菌活性。方法通过分别合成Lactoferricin B基因两条单链的部分序列,让其互为模板、引物进行PCR扩增,得到Lactoferricin B与3种变异的基因序列。将它们克隆到穿梭质粒pYES2中,构建pYES2/Lactoferricin B及3种变异基因重组质粒,转化到大肠杆菌Top10中,让其大量增殖。提取重组质粒经纯化后将其转化到S.cerevisiae中,通过营养缺陷型筛选获得重组酵母菌并通过半乳糖诱导使其表达目的蛋白。经离子交换柱纯化收集目的蛋白后,通过体外抑菌实验比较Lactoferricin B及3种变异重组蛋白的抗菌活性。结果经PCR扩增检验、DNA测序表明成功构建pYES2/Lactoferricin B与3种变异基因重组质粒。提取诱导后的蛋白进行SDS-PAGE电泳及质谱检测,证实目的蛋白的存在,相对分子质量约为3.4×103。在大肠埃希菌及金黄色葡萄球菌的抑菌实验中观测到Lactoferricin B和A17-Lactoferricin B产生了抑菌圈。结论成功构建pYES2/Lactoferricin B及变异基因重组质粒,该重组质粒能在S.cerevisiae中诱导表达目的蛋白,为进一步研究其生物功能及抗菌活性奠定了基础。

肽核酸芯片技术初探

目的 研究解决以DNA为探针的寡核苷酸芯片特异性、重复性差等问题。方法 用肽核酸 (PNA)探针制备PNA芯片 ,以荧光标记的寡核苷酸 (ODN)及HBVDNA的PCR产物为检测对象 ,探讨PNA芯片的杂交条件。用PNA芯片检测HBVDNA及单碱基突变 ,并与DNA芯片做一对比。结果 PNA探针对单碱基突变的识别力比相应的DNA探针更强 ;用其检测HBVDNA ,得到了与常规PCR试剂盒一致的检测结果。结论 PNA探针独特的结构决定了它在杂交中具有与靶DNA结合的稳定性高及特异性强等优点。

抗突变型瓜氨酸波形蛋白抗体对类风湿关节炎的诊断价值

目的:探讨抗突变型瓜氨酸波形蛋白抗体(抗MCV)对类风湿关节(rheumatoid arthritis,RA)的诊断价值。方法:收集79例RA患者、51例非RA自身免疫性疾病患者和40名健康体检者血清;采用全自动酶联免疫法定量检测抗MCV、RF IgG、RF IgA,化学发光法检测抗CCP,速率散射比浊法检测RF IgM,并对结果进行分析。结果:RA组患者血清中抗MCV含量为(451.07±40.38)IU/mL,明显高于非RA自身免疫性疾病组和正常对照组(P<0.01)。ROC曲线确定抗MCV诊断RA的最佳阈值为54.05 IU/mL;其ROC曲线下面积为0.940,较RF IgM、RF IgG、RF IgA明显增加(P<0.05),但与抗CCP比较差异无统计学意义。抗MCV诊断RA的灵敏度和特异性分别为89.87%、91.21%,高于其他4项指标;在抗MCV与其他指标联合检测中,抗MCV/抗CCP/RF IgM/RF IgG/RF IgA联合检测诊断RA的敏感性最高,为98.73%;而特异性和准确性最高的是抗MCV/抗CCP联合检测,分别为90.11%、91.18%。在RA患者中,抗MCV与抗CCP、RF IgM、RF IgG、RF IgA有相关性,其相关系数分别为0.430、0.272、0.269、0.343(P<0.05)。结论:检测血清中抗MCV水平对于筛查和辅助诊断RA具有重要意义。

家蝇抗菌肽的研究进展

从家蝇抗菌肽的分类、结构特点、作用机理、诱变、活性检验以及分子生物学研究等方面介绍了家蝇抗菌肽的研究进展。家蝇抗菌肽独特的作用机理、成熟的诱导、分离纯化技术及其相关基因的克隆、表达等研究成果均为对家蝇抗菌肽进行更深入的研究奠定了基础。采用分子生物学技术有望筛选并制备大量的高效家蝇抗菌肽。

suc2基因的克隆及在酵母基因组中的定位突变

目的 克隆酿酒酵母的蔗糖转换酶基因 (suc2 ) ,并定位突变酵母基因组中的 suc2基因以获得无蔗糖转换酶表达的酵母品系 .方法 用 PCR法从酵母 Y190基因组中扩增出suc2的成熟肽基因片段 ,克隆后测序 .将色氨酸合成酶 (TRP)基因片段插入 suc2基因中 ,构建成用于同源重组的 suc2基因定位突变载体 .导入酵母 Y190 ,用营养缺陷筛选出 suc2基因突变的克隆 .用 PCR扩增并克隆突变后的 suc2基因 ,用序列分析确定同源重组的发生 .结果 用 PCR从酵母基因组DNA中扩增出大小约 1.5 kb的 suc2基因片段 ,序列测定表明除 5 85位有一点突变 (AAC变为 AAT)外 ,所得 suc2基因与文献报道相同 ;构建的定位突变载体导入酵母细胞后 ,得到4株营养型符合的突变体 ;PCR表明这些突变体中均有 suc2基因的同源重组 ;取其中 1株的 PCR产物进行序列测定证实了同源重组的发生 .结论 克隆了正确的编码蔗糖转换酶成熟肽的 suc2基因片段 ;用同源重组方法在酵母 Y190的基因组中定位突变了