番茄黄化曲叶病毒CP基因的原核表达载体构建选择

[目的]筛选出目的蛋白能够高效表达的重组质粒。[方法]利用原核表达载体pET-32a(+)和pET-28a(+)成功构建了番茄黄化曲叶病毒(Tomato Yellow Leaf Curl Virus,TYLCV)外壳蛋白(Coat Protein,CP)基因的重组质粒p32a-CP和p28a-CP,并通过PCR和双酶切鉴定了序列连接的正确性;再分别将2个载体转化至BL21(DE3)中,采用不同浓度的IPTG对其进行诱导表达,并进行SDS-PAGE电泳检测。[结果]测序结果显示TYLCV-CP基因已定向插入p32a-CP和p28a-CP中;SDS-PAGE电泳结果显示分子量约为50 kD的目的蛋白在重组载体p32a-CP中得到了高效表达,而在重组载体p28a-CP中未表达。[结论]为下一步的抗体制备及TYLCV的免疫学检测奠定了基础。     

基于山羊痘病毒P32蛋白间接ELISA方法的建立

通过Ni柱亲和层析和G-100层析技术对真核表达蛋白进行纯化,SDS-PAGE分析GPVP32蛋白纯化效果;应用琼脂扩散试验检测纯化蛋白的抗原反应性,并以纯化蛋白作为包被抗原建立间接ELISA方法。结果显示,从真核表达产物中获得了纯化的GPVP32蛋白,琼脂扩散试验显示纯化的GPVP32蛋白具有良好的抗原反应性;基于纯化蛋白建立的间接ELISA方法具有良好的特异性、敏感性和重复性。结果表明,本试验建立的间接ELISA方法可用于山羊痘流行病学的调查和血清抗体水平的监测,为山羊痘的防控提供了有效技术。     

闽南山地DH32-29桉树无性系的耐寒性调查分析

通过对华安葛山林场较高海拔山地种植的DH32-29桉树无性系的寒害进行调查,结果表明:幼龄林分寒害较重,林龄较大的林分寒害相对较轻;坡向、坡位、地形地势等是影响寒害程度的外在因素。在本区域较高海拔山地种植桉树应选择避寒环境,避免在盆地、洼地、坡地下坡和山谷等处种植;根据实践经验提出搞好林分抚育、冬前增施钾肥不施氮肥可以减轻寒害、合理搭配桉树品种、选择抗寒能力强的品种是避免寒害的根本措施。     

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla_TEM-52 gene and two of them also harboured bla_TEM-1b. The bla_CTX-M-14 gene was identified in three isolates (in association with bla_TEM-1b in one of them), and bla_CTX-M-32 was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58(C → T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim–sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.     

羊痘病毒P32蛋白的研究进展

P32蛋白是所有羊痘病毒具有的结构蛋白之一,含有重要的免疫原性决定簇。P32蛋白既是致病的关键因素,也是激发机体产生抗体并产生保护性免疫的主要成分。作者就羊痘病毒P32蛋白的基因组结构、抗原表位及P32蛋白应用于羊痘病毒诊断和免疫方面的研究进展作一综述。     

聚天门冬氨酸同源多肽的水稻产量效应和磷素营养吸收效应研究

聚天门冬氨酸同源多肽对水稻产量效应研究表明,聚天门冬氨酸同源多肽对水稻秧苗生长和根的生长有较大的促进作用,根的鲜重、干重和根长分别比清水对照增加28.2%、46.2%、17.9%,对水稻经济性状及产量也有较好的作用,有效穗数、实粒数、千粒重、考种产量和实际产量分别比清水对照增加14.21%、13.44%、2.38%、16.80%、7.74%。利用32P同位素示踪试验研究了聚天门冬氨酸同源多肽对水稻磷素营养吸收的效应,发现喷施聚天门冬氨酸同源多肽的初期对水稻吸收肥料磷有抑制作用。随着时间的推移,聚天门冬氨酸同源多肽本身含有的速效磷被完全吸收后,聚天门冬氨酸同源多肽的富集作用逐渐表现出来,从而表现为20 d时处理的水稻对磷素的吸收略有增加。30 d时,由于聚天门冬氨酸同源多肽逐渐被微生物降解,大量释放出被固定的磷素,表现为测量时的结果比对照略有降低。     

抗除草剂水稻新品系“E32HR”的选育

以基因枪法转化获得的转bar基因水稻"HR"为供体亲本,两系法亚种间杂交稻优势组合"65396"的恢复系"E32"为受体亲本,经过4次回交转育成对除草剂Basta具有稳定抗性的水稻新品系"E32HR"。对"E32HR"及其杂种"培矮64S/E32HR"的主要农艺性状进行比较分析的结果表明,除了"E32HR"的株高明显高于"E32"以外,其余性状均无明显差异。文中对转基因育种与常规育种的结合以及抗除草剂水稻的利用等问题进行了讨论。     

山羊痘病毒P32基因在杆状病毒中的表达

采用PCR亚克隆了山羊痘病毒P32基因片段,将其插入杆状病毒转座载体质粒pFastBacⅠ中,构建了重组质粒pFastBac-P32;再将该质粒转化DH10 Bac感受态细菌,进行体内重组,然后经三重抗性及蓝白斑筛选,获得杆状病毒重组质粒Bacmid-P32;将Bacmid-P32转染Sf9细胞,获得重组杆状病毒;最后用SDS-PAGE和Western-blotting对P32基因的表达进行检测.结果表明,P32基因在重组杆状病毒中获得表达,且表达产物可以被多抗所识别.     

Effects of DARPP-32 on the drug sensitivity of SH-SY5Y cells

AIM: To investigate the effects of DARPP-32 on the drug sensitivity of human neuroblastoma cell line SH-SY5Y. METHODS: The plasmid containing cDNA of DARPP-32 gene and the small interfering RNA eukaryotic expression vector specific to human DARPP-32 gene were constructed and transfected into SH-SY5Y cells. Stable transfectants were identified by RT-PCR and Western blotting analysis. MTT assay and flow cytometry assay were used to investigate the effects of DARPP-32 on the drug sensitivity and the drug accumulation in cell models. The expressions of Bcl-2，Bax, P-gp and MRP were analyzed by RT-PCR and Western blotting. RESULTS: The stable clones with increased or decreased DARPP-32 expression were successfully established. Up-regulation of DARPP-32 significantly enhanced the drug sensitivity and the drug accumulation of SH-SY5Y cells due to down-regulation of P-gp and Bcl-2 expressions. Down-regulation of DARPP-32 significantly reduced the drug sensitivity of SH-SY5Y cells and decreased drug accumulation in SH-SY5Y cells. CONCLUSION: DARPP-32 might mediate the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents.