Production and characterization of a single-chain Fv antibody–alkaline phosphatase fusion protein specific for ampicillin

Anti-AMP scFv were prepared by cloning variable region genes (VH and VL) from hybridoma cell line which secretes antibodies against AMP, and phoA gene was cloned from Escherichia coli strain K12 chromosomal DNA. The resulting scFv and phoA gene were inserted into the expression vector pBV220 to express the scFv–AP fusion protein in E. coli strain BL21. The puri?ed scFv-AP fusion protein was used to develop an ic-CLEIA method for detection of AMP. The limit of detection (IC₁₅) was 0.11?±?0.004?ng/mL and the IC₅₀ was 0.75?±?0.04?ng/mL, respectively. The assay was 3 times as sensitive as the corresponding ic-ELISA based on the same fusion protein. IC-CLEIA was used to analyse AMP spiked milk samples, and the validation was con?rmed by HPLC. Good correlation between ic-CLEIA and HPLC data was obtained (R^2?=^?0.9934). It indicated that the developed method can apply to effectively monitor food safety.     

PRRX‐NCOA1/2 rearrangement characterizes a distinctive fibroblastic neoplasm

Fibroblastic/myofibroblastic neoplasms represent a broad, and occasionally diagnostically challenging, category of soft tissue neoplasms. A subset of these tumors defy conventional classification. However, with the advent of next‐generation sequencing, the identification of disease‐defining molecular alterations is gradually improving their subclassification. Following identification of two index cases of a distinctive fibroblastic neoplasm with a fusion gene involving PRRX1 and NCOA1, we performed a retrospective review to further characterize this entity. We identified two additional cases, including one with a fusion between PRRX1 and NCOA2. The average patient age was 38 years, and three patients were female. Two tumors occurred on the neck, and the others involved the groin and thigh. Tumors were centered in the subcutis and ranged from 2.3 to 14.0 cm (average 5.8 cm). Morphologically, they were predominantly hypocellular, with focal hypercellularity. They were composed of monomorphic spindle‐stellate cells with a vague fascicular pattern. The nuclei were bland with only rare mitotic activity, and occasional multinucleation. The intervening stroma was typically abundant and ranged from myxoid to collagenous, with frequent rope‐like collagen bundles. Three of the cases had a prominent vasculature ranging from numerous small curvilinear vessels to ectatic and branching staghorn‐like vessels. Immunohistochemistry was negative for desmin, smooth muscle actin, S100, CD34, keratin, and epithelial membrane antigen. Each of the patients was treated by simple excision and none of the tumors were associated with local recurrence or metastasis. Based on their unique morphological and molecular attributes, we believe this represents a novel fibroblastic tumor for which we have tentatively proposed the name “PRRX‐NCOAx‐rearranged fibroblastic tumor.”     

Emerging soft tissue tumors with kinase fusions: An overview of the recent literature with an emphasis on diagnostic criteria

A recent breakthrough in the classification of soft tissue tumors (STT) has been a significant expansion in the number of neoplasms associated with NTRK and other kinase related fusions. This is important not only for diagnostic purposes, but also because it opens new avenues for targeted therapy. Indeed, recent clincal trials have shown significant benefit across multiple tumor‐types, prompting approval of NTRK inhibitors for clinical use in the setting of advanced/metastatic NTRK‐rearranged neoplasms. Despite these therapeutic oportunities, diagnostic challenges have transpired in recognizing these emerging new histologic subtypes of kinase fusions positive‐STT prospectively. This, in part, is attributable to their wide morphologic spectrum, variable risk of malignancy, and non‐specific immunoprofile. As such, recommendations for pathologic criteria and immunohistochemical testing are needed to improve classification and streamline the small subset of potential candidates for further molecular validation. This overview summarizes the key histologic features of various STT associated with NTRK and other kinase fusions, which appear to share a similar morphologic spectrum. Immunohistochemically, many of these tumors, regardless of the kinase fusion type, notably show co‐expression of S100 and CD34; issues related to the utility of pan‐NTRK and NTRK1 immunostaining are therefore summarized. Finally, I discuss the role of confirmatory molecular testing and how, in some instances, this may also be of prognostic value. This review is intended as a critical summary of the current literature to emphasize pathologic criteria for improving recognition of this emerging and complex group of kinase fusion associated STT.     

单侧双通道内镜下经椎间孔腰椎椎间融合术的研究进展

单侧双通道内镜下腰椎椎间融合术是采用双通道在持续冲洗及内镜直视下行腰椎椎体间减压及融合的手术,具有手术器械简单、操作灵活性强、创伤小、学习曲线相对平缓等优势,常应用于腰椎间盘突出、椎管狭窄及腰椎滑脱等腰椎退行性疾病。随着该术式在脊柱疾病中的广泛应用,一些潜在的优势及不足也逐渐被发现。单侧双通道内镜下腰椎椎间融合术的手术入路有两种：后路腰椎椎体间融合术（PLIF）和经椎间孔腰椎椎体间融合术（TLIF）。目前研究较多的是TLIF,本文就单侧双通道内镜下经椎间孔腰椎椎间融合手术在腰椎退行性疾病中的临床疗效进行阐述。     

β2M-绿色荧光蛋白在MHC-Ⅰ肽亲和力实验中的应用

目的 构建并表达β2微球蛋白-绿色荧光蛋白融合蛋白,并应用该蛋白建立一种全新的MHC-Ⅰ肽亲和力实验方法.方法 构建β2M-ZsGreen-6×His标签的融合蛋白真核表达质粒,转染293FT细胞表达,并以镍亲和层析法纯化,将该融合蛋白与表位肽共同与,12细胞反应,通过流式细胞术检测T2细胞标记绿色荧光的情况鉴定表位.结果 构建了B2微球蛋白-绿色荧光蛋白真核表达质粒并表达、纯化得到融合蛋白,该蛋白可应用于亲和力实验,进行表位鉴定.结论 β2微球蛋白-绿色荧光融合蛋白应用于亲和力实验鉴定CTL表位方法可行,步骤简便,适用于大通量表位鉴定.     

微创单侧TLIF辅助单边钉棒固定治疗腰椎间盘病变的初步报告

目的探讨采用微创经腰椎间孔椎体间融合术（TLIF）辅助单边钉捧固定治疗腰椎间盘病变的适应证、手术方法及初步疗效。方法自2006年1月至2008年6月,15例腰椎间盘病变患者进行微创TLIF治疗。其中,男性7例,女性8例：年龄48～64岁,平均年龄54．7岁．病程1．5～3．0年,平均2年。其中L3-43例L4-5,8例,L5-S17例：单节段病变12例．双节段病变3例．术前视觉模拟评分法（VAS）评分为（7．6±1．0）分,欧氏失能指数（ODI）评分为（42．4±2．7）分．Prolo功能评分为（10．5±1．6）分,、所有患者均有下腰痛．仅有单侧下肢痛、麻木。结果手术时间120～240min,统计数据（182．3±35．5）min。术中出血150．420mL．统计数据（280．7±81．2）mL。住院天数7～35d．统计数据（17．7±6．2）d。所有病例随访时间8～21个月,平均随访时间15．2个月。术后VAS评分（3．0±0．8）分,ODI评分（16．4±2．9）分．改良Prolo评分（17．0±0．9）分,与术前比较差异有统计学意义（P〈0．05）,其中优9例,良4例,可2例,总优良率86．7％。该组15例18个融合节段,术后6个月三维CT重建,有15个节段融合,融合率833％。结论微创TLIF辅助单边钉棒系统同定具有软组织损伤小、出血量少、不破坏对侧的正常结构、住院时间短、功能恢复好、并发症发生率低的优点。但应严格掌握适应证．长期结果还需进一步随访,     

人GST-PTEN融合蛋白的原核表达和纯化

目的构建人第10号染色体上磷酸酶和张力蛋白同源缺失的基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)原核表达质粒,使其在原核细胞中高效表达并进行纯化。方法将全长片段插入原核表达载体pGEX-4T-1,构建重组子pGEX-4T-1-PTEN,转化BL-21感受态细胞。经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达PTEN融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western印迹分析证实蛋白表达的特异性。并用谷胱甘肽-S-转移酶(glutathione-Stransferase,GST)亲和层析对融合蛋白进行纯化。结果成功构建了原核表达载体pGEX-4T-1-PTEN,并将PTEN融合蛋白的成功表达,通过SDS-PAGE电泳、Western印迹分析,证实了蛋白表达的特异性。并对蛋白进行了纯化,获得了GST-PTEN融合蛋白的纯品。结论成功表达、纯化了GST-PTEN融合蛋白,为进一步研究PTEN蛋白的功能及其与其它功能性蛋白的相互作用研究奠定了基础。     

脊柱融合内固定致邻近节段退变的生物力学机制

目的：从生物力学角度了解融合内固定对腰椎邻近节段的影响。方法：9具新鲜腰1～骶椎尸体标本分别近、远端固定，在脊柱三维运动实验机上施以8Nm纯力偶矩模拟人体行屈伸、左右侧弯及旋转活动，观察L3～4、L4～5、L5～Sl节段运动范围(ROM)；随后进行各种模拟手术并安装内固定，依次测定CD内固定(CD)、CD加椎体间植骨(CD-骨块)、CD加TFC(CD—TFC)状态下各节段ROM值并进行统计学分析。结果：①各融合术式均可使固定节段达到即刻稳定性，L4～5节段ROM值明显减小；②L3～4节段在屈伸、侧弯和旋转时有明显位移增加，L5S1节段在旋转时ROM值明显增加，屈伸和侧弯时ROM值改变无统计学意义。结论：脊柱融合内固定术后，相邻节段位移增加、运动模式改变，易继发不稳和退变。     

细粒棘球绦虫疫苗候选分子EgA31的GST融合蛋白原核表达及纯化

目的 :构建细粒棘球绦虫疫苗候选分子EgA31重组质粒 ,表达及纯化EgA31 GST融合蛋白 ,为疫苗的研究奠定基础。方法 :PCR扩增EgA31cDNA ,将得到的cDNA经限制性内切酶酶切 ,然后亚克隆入pGEX 5X 3质粒 ,转化BL2 1宿主菌 ,IPTG诱导重组质粒的表达 ,亲和层析纯化表达产物 ,Bradford法测定重组蛋白含量 ,用SDS PAGE和Westernblot进行分析鉴定。结果 :SDS PAGE显示分离纯化的EgA31 GST融合蛋白为 4 5kDa ,测序检验重组质粒中EgA31cDNA序列正确。EgA31 GST融合蛋白免疫豚鼠得到的抗血清可与细粒棘球绦虫原头蚴总蛋白在 6 6kDa处特异性反应。结论 :EgA31 GST融合蛋白获得高效表达 ,并成功纯化 ,初步实验证明具有良好的免疫原性 ,可用于进一步疫苗的免疫注射实验     

白血病基因诊断及其信息系统建立

目的 用基因诊断技术对白血病分型诊断及微小残留病（MRD）监控,同时对所检测数据建立信息系统.方法 巢式RT-PCR扩增急髓性白血病融合基因,PCR扩增急性淋巴细胞白血病基因重排,用Delphi程序设计信息系统.结果 M2b型白血病能扩增出AML1/ETO融合基因;M3型白血病能检测出L型和S型融合基因;慢粒白血病可检测出b3a2和b3a2融合基因;急淋白血病可检测出IgH、TCRγ、TCRδ基因重排.结论 基因诊断能有效进行白血病分型及MRD监控,信息系统的建立方便了检测信息的管理及查询.