The clone, prokaryotic expression of cry2Ab30 gene and its insecticidal mechanism against Plutella xylostella L.

Cry毒素是苏云金芽胞杆菌（Bt）产生的δ内毒素之一，其特征是对多种农业害虫均具有杀虫毒性，被广泛应用于微生物农药及转基因植物中。 本研究以福建省农科院分离保存的高毒力Bt菌株FJAT-12为模板，克隆得到一个新的cry2Ab基因，多序列比对结果显示该cry2Ab基因有2个保守性位点发生突变，为第四类新基因，并被命名为cry2Ab30。构建pET30a-cry2Ab30原核表达载体，表达Cry2Ab30蛋白并利用WesternBlot进行验证；分别从诱导剂IPTG浓度、转数和温度三个方面对原核表达条件进行优化，我们发现Cry2Ab30最佳的表达条件为：诱导剂IPTG浓度0.2mmol/L、转...Cry toxin, a kind of δ-endotoxin produced by Bacillus thuringiensis (Bt), was characterized by its insecticidal activity among agricultural pests and was widely used in microbial pesticide and transgenic plants. In present study, a new cry2Ab gene was cloned from the high-toxicity Bt strains FJAT-12 that was isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science....学位：理学硕士院系专业：生命科学学院_生物化学与分子生物学学号：2162012115235

Effectiveness of total knee arthroplasty using three-dimensional printing technology for knee osteoarthritis accompanied with extra-articular deformity

目的探讨3D打印技术辅助人工全膝关节置换术(total knee arthroplasty,TKA)治疗合并关节外畸形的膝骨关节炎(knee; osteoarthritis,KOA)的临床疗效。方法2013年3月2015年12月,收治15例(18膝)合并关节外畸形的KOA患者。男6例(6; 膝),女9例(12膝);年龄55~70岁,平均60.2岁;病程7~15年,平均10.8年。单膝12例,双膝3例。膝关节学会评分系统(KSS)临床; 评分为(57.441.06)分,功能评分为(60.881.26)分。膝关节活动度为(72.220.18)°。下肢力线偏移(18.890.92)°; 。合并股骨侧畸形8例(10膝),胫骨侧畸形5例(5膝),股骨侧及胫骨侧畸形2例(3膝)。术前3D打印骨骼模型、截骨导航模板并设计手术方案,选择合; 适的膝关节假体后实施TKA。结果手术时间65~100 min,平均75.6 min;术中出血量50~150 mL,平均90.2; mL。术后均未出现切口愈合不良、感染、血栓等并发症。患者均获随访,随访时间12~30个月,平均22个月。末次随访时,X线片示假体位置均良好,未发; 现松动、下沉;下肢力线偏移(2.000.29)°,与术前比较差异有统计学意义(t=13.120,P=0.007);KSS临床评分为(87.500; .88)分、功能评分为(81.941.41)分,与术前比较差异有统计学意义(t=27.553,P=0.000;t=35.551,P=0.000); ;膝关节活动度为(101.941.42)°,与术前比较差异有统计学意义(t=31.633,P=0.000)。结论对于合并关节外畸形的KOA,采用; 3D打印技术辅助TKA,可达到个体化治疗、降低手术难度,有效矫正畸形,恢复患者膝关节功能的目的。Objective To evaluate the effectiveness of total knee arthroplasty (TKA); using three-dimensional (3D) printing technology for knee osteoarthritis; (KOA) accompanied with extra-articular deformity. Methods Between March; 2013 and December 2015, 15 patients (18 knees) with extra-articular; deformity and KOA underwent TKA. There were 6 males (6 knees) and 9; females (12 knees), aged 55-70 years (mean, 60.2 years). The mean; disease duration was 10.8 years (range, 7-15 years). The unilateral knee; was involved in 12 cases and bilateral knees in 3 cases. The clinical; score was 57.441.06 and the functional score was 60.881.26 of Knee; Society Score (KSS). The range of motion of the knee joint was; (72.220.18)°. The deviation of mechanical axis of lower limb was; (18.890.92)° preoperatively. There were 8 cases (10 knees) with; extra-articular femoral deformity, 5 cases (5 knees) with; extra-articular tibial deformity, and 2 cases (3 knees) with; extra-articular femoral and tibial deformities. Bone models and the; navigation templates were printed and the operation plans were designed; using 3D printing technology. The right knee joint prostheses were; chosen. Results The operation time was 65-100 minutes (mean, 75.6; minutes). The bleeding volume was 50-150 mL (mean, 90.2 mL). There was; no poor incision healing, infection, or deep venous thrombosis after; operation. All patients were followed up 12- 30 months (mean, 22; months). Prostheses were located in the right place, and no sign of; loosening or subsidence was observed by X-ray examination. At last; follow-up, the deviation of mechanical axis of lower limb was; (2.000.29)°, showing significant difference when compared with; preoperative one (t=13.120, P=0.007). The KSS clinical score was; 87.500.88 and function score was 81.941.41, showing significant; differences when compared with preoperative ones (t=27.553, P=0.000;; t=35.551, P=0.000). The range of motion of knee was (101.941.42)°,; showing significant difference when compared with preoperative one; (t=31.633, P=0.000). Conclusion For KOA accompanied with extra-articular; deformity, TKA using 3D printing technology has advantages such as; individualized treatment, reducing the difficulty of operation, and; achieving the satisfactory function

Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera:Plutellidae)

【目的】克隆和表达小菜蛾PluTEllA XylOSTEllA氨肽酶基因,并进行基因序列分析和同源建模分析。【方法】以小菜蛾中肠C dnA为模板克隆分析氨肽酶基因序列,原核表达氨肽酶蛋白并进行酶活性测定,应用配体印迹分析氨肽酶与Cry2Ab蛋白的结合,通过蛋白质建模对突变位点进行分析。【结果】从小菜蛾中肠C dnA扩增出氨肽酶基因,该基因全长2 853 bP,编码950个氨基酸,预测蛋白分子量为107.3871 k dA,等电点为5.24;进化树分析显示,克隆得到的氨肽酶基因属于APn家族5,将其命名为PX APn5(gEn bAnk登录号:kM034756)。PX APn5蛋白具有鳞翅目昆虫氨肽酶蛋白的保守性特征,即含有n-糖基化位点、O-糖基化位点和gPI锚定位点,具有“HEXXH“锌蛋白酶结构域和C端跨膜区域。在大肠杆菌ESCHErICHIA COlI中原核表达PX APn5,表达产物经SdS-PAgE电泳,在110 k dA附近出现特异性条带;酶活性测试显示菌体破碎上清液具有氨肽酶活性,比活力为1 047.2 u/g。配体印迹结果显示表达的PX APn5能与Cry2Ab蛋白特异性结合。多序列比对结果表明,与其他已报道的小菜蛾氨肽酶相比,PX APn5氨基酸序列有3个保守性位点发生了突变,并通过蛋白质建模的方式表征突变位点。【结论】本研究成功克隆和表达了具有氨肽酶活性的小菜蛾氨肽酶,并发现其能与Cry2Ab蛋白特异性结合;通过蛋白质建模对氨肽酶突变位点的特征及功能进行了预测。这些结果对小菜蛾氨肽酶的功能性研究提供了理论基础。【Aim】The aim of this study is to analyze the cloning,expression and homology modeling of midgut aminopeptidase gene Px APN5 in the diamondback moth,Plutella xylostella.【Methods 】 A aminopeptidase( APN) gene was cloned from the P.xylostella midgut,and bioinformatics analysis of the gene was performed.The APN protein was expressed using prokaryotic expression system,and its enzymatic activity was assayed.The interaction between APN and Cry2 Ab was determined by using ligand blot analysis.Homology modeling of APN gene was conducted for the prediction of characteristics and functions of mutation sites.【Results】The sequencing results showed that the cloned APN gene( NCBI accession no.: KM034756) is 2 853 bp in length and encodes 950 amino acids with the predicted molecular weight of 107.3871 k Da and isoelectric point of 5.24.Phylogenetic tree analysis indicated that this APN gene belongs to class 5 of APN family,and it was named as Px APN5.Px APN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and Oglycosylation sites,GPI anchor point,C-transmembrane domains and zinc-metalloprotease domain(361HEXXH365).A 110 k Da specific protein band appeared when APN protein was inducibly expressed in Escherichia coli.Aminopeptidase activity assay showed that the supernatant of broken bacteria possessedaminopeptidase activity and its specific activity was 1 047.2 U / g.The ligand blot result indicated that Px APN5 could bind to Cry2 Ab specially.Multiple alignment of amino acid sequences demonstrated that there are three mutations in Px APN5 when compared to other known APN proteins from P.xylostella.【Conclusion】The Px APN5 protein with aminopeptidase activity was successfully cloned and expressed,and it could bind to Cry2 Ab.Prediction of characteristics and functions of mutation sites in Px APN5 was carried out by homology modeling.These results laid the foundation for the functional research of Px APN.国家自然科学基金项目(31371999;31370059

小鼠端粒酶蛋白亚单位重组腺病毒载体的构建及鉴定

【目的】构建小鼠端粒酶蛋白亚单位（mouse telomerase reverse transcriptase，mTERT）基因重组腺病毒载体，为下一步研究其体外表达和动物实验研究提供基础。【方法】从肝癌细胞中提取总RNA。采用RT-PER技术扩增mTERT的基因编码区序列，将序列定向克隆至真核表达载体pAC，将此表达质粒与腺病毒重组质粒pJM17共同转染293细胞，经同源重组产生重组腺病毒载体Ad-mTERT，纯化后的重组腺病毒载体Ad-mTERT在293细胞大量扩增并通过氯化铯密度梯度离心法纯化，测定病毒滴度。【结果】PCR及酶切证实：mTERTDNA正确克隆到穿梭质粒pAC中，带mTERTDNA的表达盒成功重组到腺病毒载体基因组E1A缺失区，并在293细胞中成功包装出具有感染活性的重组腺病毒Ad-mTERT。【结论】成功构建了小鼠TERT基因重组腺病毒载体，为研究其用于肿瘤的基因治疗奠定基础

小菜蛾钙黏蛋白PxCR结构域的克隆、原核表达及功能分析

钙黏蛋白（cadherin）是一类跨膜糖蛋白,因其在苏云金芽胞杆菌（Bacillus thuringiensis,Bt）的杀虫过程中作为主要的受体而受到广泛研究.钙黏蛋白的特征之一是由若干钙黏蛋白重复单元（cadherin repeats,CR）组成的长链状蛋白,其中靠近细胞膜的CR结构域被认为是钙黏蛋白与Bt毒素发生互作的区域.小菜蛾（Plutella xylostella）钙黏蛋白由11个CR结构域和1个跨膜结构域组成,其中第10和11个CR结构域PxCR被认为是钙黏蛋白与Bt毒素的互作区域.本研究从小菜蛾中肠cDNA中克隆得到小菜蛾钙黏蛋白PxCR结构域的DNA片段,并通过原核表达系统对PxCR结构域进行表达.配体印迹结果表明PxCR可以特异性和Cry2Ab结合;杀虫实验结果表明PxCR能提高Cry2Ab对小菜蛾的毒力.此外,利用蛋白质同源建模和糖基化位点预测网站对PxCR蛋白质结构进行了分析.上述结果表明PxCR可能参与了Cry2Ab毒素对小菜蛾的毒杀过程,为后续研究小菜蛾钙黏蛋白和Cry2Ab的相互作用机制奠定了基础.国家自然科学基金(31371999；31370059)