Expression of fusion protein anti-HER2-ScFv-GFP in insect cells and binding to surface of breast cancer cells

AIM: To express a recombinant fusion protein anti-human epidermal growth factor receptor-2 single-chain variable fragment with green fluorescent protein (anti-HER2-ScFv-GFP) using the insect cells-Bac-to-Bac baculovirus expression system and to analyze the binding function of this fusion protein with HER2 on the surface of the breast cancer cells. METHODS: Human anti-HER2-ScFv gene from mice was fused with GFP gene. To obtain the recombinant plasmid pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP, we inserted it into Bac-to-Bac baculovirus expression plasmid pFAST Bac-to-Bac HT A. The identified recombinant plasmid was transferred into Escherichia coli DH10Bac to allow the generation of a recombinant bacmid. After transfected the recombinant virus bacmid into the insect cells Tn-5B1-4, the recombinant virus was collected to infect Tn-5B1-4. SDS-PAGE and Western blotting analysis were used to verify the expression product in Tn-5B1-4. The fusion protein was purified with Ni²⁺-NTA affinity chromatography. The purified fusion protein was bound to the surface of HER2-positive breast cancer cells SKBR3 and HER2-negative breast cancer cells MCF7. The binding effects on the surface of breast cancer cells were observed under laser confocal microscope. RESULTS: The fusion gene anti-HER2-ScFv-GFP was successfully constructed with the length of 1 539 bp. The green fluorescence was also observed in Tn-5B1-4 cells infected with the recombinant virus under fluorescent microscope. A 60 kD protein was examined and confirmed by SDS-PAGE and Western blotting. Under laser confocal microscope, strong green fluorescence was observed on the surface of the HER2-positive breast cancer cells SKBR3. However, no green fluorescence was observed on the surface of HER2-negative breast cancer cell MCF7. Obvious green fluorescence on the surface of HER2-positive breast cancer cell SKBR3 was also found after the cells were eluted with 1×PBS. CONCLUSION: The fusion protein anti-HER2-ScFv-GFP was successfully expressed in insect cells Tn-5B1-4, and can firmly bind to the surface of breast cancer cells SKBR3 and emit the green fluorescent light.     

基于非抽样Shearlet变换的红外与可见光图像融合方法

针对同一场景红外图像与可见光图像的融合问题,提出了一种基于非抽样Shearlet变换(NSST)的融合算法。首先对源图像进行多尺度、多方向NSST分解,得到低频子带系数和各带通方向子带系数;然后,在局部区域结构相似度的基础上,采用基于局部区域能量的方法选择融合图像的低频子带系数;基于脉冲耦合神经网络(PCNN)对带通方向子带空间频率(SF)的响应而得到的点火次数选择融合图像的带通方向子带系数,得到融合图像的NSST系数;最后经过非抽样Shearlet逆变换得到融合图像。实验结果表明:与其他5种相关的融合方法相比,该方法可获得具有更好视觉效果和更优量化指标的融合图像。     

农机农艺融合破解辣椒收获机技术难点

农机与农艺相结合是一条适应当前农业机械研发的道路。该文通过对辣椒收获机研发现状及研究成果的分析总结,验证了农机农艺相结合是适合农机今后发展,并值得进一步在其他农机研发上推广借鉴的有效途径。      

CHX-GFP融合基因的构建及在胡萝卜愈伤组织中瞬时表达

为构建CHX-GFP融合基因并将其在胡萝卜愈伤组织中瞬时表达。采用融合PCR方法,将属于CAP2家族的Na+/H+反向转运蛋白基因CHX和GFP基因相融合,利用中间载体,采用DNA重组技术将CHX-GFP融合基因插入到p1301植物表达载体中,采用冻融法将重组质粒导入到农杆菌中,遗传转化时所用侵染液为1/2MS+AS 100μmol/L,共培养基为MS+6-BA 0.5 mg/L+2,4-D 0.5 mg/L+琼脂6 g/L+蔗糖30 g/L+葡萄糖10 g/L+AS 50μmol/L,共培养时间为4 d。成功构建了p1301-35S-CHX-GFP-Nos植物表达载体,CHX-GFP融合基因在胡萝卜愈伤组织中得到瞬时表达,表达效率为75%。采用融合PCR获得CHX-GFP融合基因的方法比较简单,无须知道DNA序列的酶切位点,省时省力,CHX-GFP融合基因及其表达载体的成功构建将为进一步探查CHX亚细胞定位及CHX基因的功能奠定了基础。     

Contruction of the Genetic Engineering Strain Expressed Nontoxic ST1-LTB Fusion Protein Against Enterotoxigenic Eschenichia coli

Thermostable enterotoxinⅠ(ST1)mutant genes and thermolabile enterotoxin B subunit(LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902.The recombinant expression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3).The ST1-LTB fusion protein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB)and the fusion protein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.More important,mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro.These sera antibodies were able to neutralize the biological activity of native ST1 in the suckling mouse assay.Hence the ST1-LTB fusion protein was nontoxic and immunogenic,the constructed recombinant strain BL21(DE3)(pZST3LTB)could be used as a candidate of vaccine strain.     

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

融合蛋白mCry1AbVip3A对亚洲玉米螟和草地贪夜蛾的抗虫效率分析

将融合基因mCry1AbWp3A构建原核表达载体pET30A-mCry1AbVip3A,转入大肠杆菌BL21(DE3),通过优化诱导条件获得mCry1AbVip3A蛋白.室内生测mCry1AbVip3A蛋白对亚洲玉米螟和草地贪夜蛾的杀虫效果,发现饲喂终浓度为128.44 ng/mL的mCry1AbVip3A蛋白的亚洲玉...     

产肠毒素大肠杆菌K88ac-STⅡ-LTA2/LTB融合基因在苜蓿中的表达

为提高农杆菌介导的苜蓿遗传转化效率,获得转K88ac-STⅡ-LTA2/LTB基因苜蓿植株,以苜蓿下胚轴为外植体,对影响遗传转化的预培养时间、农杆菌菌液浓度、筛选方式等进行研究.结果表明,苜蓿下胚轴预培养3 d,菌液浓度为OD6000.5,筛选剂Kan浓度第一阶段和第二阶段分别为30和50 mg/L ,延迟筛选7 d时转化效率最高.对转基因苜蓿进行PCR和PT-PCR检测,初步证实目的基因已经整合到苜蓿基因组中,转化率达28.4%.     

绵羊肺炎支原体Y98标准株P30外膜蛋白与IFN-γ的融合表达及其免疫原性

以绵羊肺炎支原体(Mycoplasma oumvipneonia,MO)标准株Y98的P30外膜蛋白为抗原,同时以细胞因子IFN-γ为佐剂,将两者串联融合表达.小鼠免疫保护试验结果显示,P30重组蛋白的免疫保护率为60％,IFN-γ重组蛋白的免疫保护率为20％,P30-IFN-γ重组蛋白的免疫保护率达到80％.间接ELISA试验证实,经100 μL与200 μL P30-IFN-γ重组蛋白免疫小鼠的血清中,P30抗体效价分别为1:32 000与1:64 000.以上结果证实,所制备的重组蛋白对小鼠实验个体基本安全,且P30-IFN-γ重组蛋白中的细胞因子IFN γ组分可以调节机体免疫机能,增强P30蛋白的免疫保护效果.     

花生白藜芦醇合成酶基因PNRS1的克隆及其在原核中的表达

采用RT-PCR克隆花生白藜芦醇合成酶(resveratrol synthetic enzyme, RS)基因全长,命名为PNRS1,GenBank登录号为FM955393。序列分析表明该基因的开放读码框1 170 bp,编码389个氨基酸残基。以花生品种鲁花14基因组DNA为模板经PCR扩增,获得该基因的基因组序列长1 537 bp,包含2个外显子和1个内含子。比较发现,PNRS1与其他已知RS序列的同源性达91%~95%。表达模式分析显示,PNRS1在花生根中特异表达,并可被紫外线UV-B诱导。PNRS1与pET-28a(+)构建原核表达载体,经IPTG诱导后可表达获得相对分子量约为46 kD的外源融合蛋白。以上结果证实PNRS1属花生RS基因家族成员,并为进一步分析该基因的功能奠定了基础。