Effect of 3 isoforms of Ikaros on proliferation of human ovarian cancer SKOV3 cells

AIM: To investigate the effect of Ikaros isoforms on the proliferation of human ovarian cancer SKOV3 cells. METHODS: Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV3 cells. The cell cycle was analyzed by flow cytometry. The cell cycle-related proteins were detected by Western blot. RESULTS: IK1 and IK2 expression inhibited SKOV3 cells proliferation. Flow cytometry analysis indicated that IK1 and IK2 induced SKOV3 cell cycle arrest at the G₁ phase. IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV3 cells. Compared with control EV group, IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D1 and cyclin D2, which did not change in IK6 group. CONCLUSION: IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G₁ phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV3 cells.     

重叠区扩增法合成抗菌肽B基因

人工设计并合成了抗菌肽 B基因的 4个寡聚核苷酸片段 ,通过重叠区扩增法 ,扩增出了相当于抗菌肽基因全长的寡聚核苷酸片段。经克隆测序 ,证明成功地实现了抗菌肽基因的人工合成、拼接和克隆。     

Comparative sequence analysis of four myosin heavy chain isoforms expressed in porcine skeletal muscles: Sequencing and characterization of the porcine myosin heavy chain slow isoform

A nucleotide sequence including the full coding region for the myosin heavy chain (MyHC) slow isoform was determined from the longissimus skeletal muscle. The deduced amino acid sequence was 1935 residues, which was the same length as the human and rat MyHC-slow isoforms. The porcine MyHC-slow isoform showed 97.6% and 97.4% amino acid identities to the human and rat isoforms on their entire regions, respectively. The functional regions were also highly conserved among mammalian MyHC-slow isoforms. Amino acid substitutions between the porcine MyHC-slow and MyHC-fast isoforms were concentrated on the functional regions. Loop 1, the controlling region of nucleotide binding and release, was conserved among the fast isoforms, but not between the slow and fast isoforms. Loop 2, a part of the actin binding region, was not conserved among any of the isoform types, and the most substitutions in this region were found in the slow isoform. The myosin essential light chain binding region was conserved among the fast isoforms, except for some substitutions in the 2b isoform, but was clearly different in the slow isoform. The myosin regulatory light chain binding region was conserved among the fast isoforms, but not between the slow and fast isoforms. These results indicate that the functional difference between the porcine MyHC-slow and -fast isoforms are controlled by the sequence diversity at the four functional regions compared.     

不同温度下水稻胚乳AGPase各同工酶基因表达特征分析(英文)

[目的]分析不同温度对水稻胚乳中AGPase各同工酶基因表达水平的影响。[方法]以特青和泰国香米为材料,利用人工气候箱设置高温(日平均温度33℃)和适温(日平均温度25℃)2个温度处理,结合实时荧光定量PCR技术分析比较了水稻淀粉合成关键酶腺嘌呤-葡萄糖焦磷酸化酶(AGPase)7个同工酶基因AGPS1、AGPS2a、AGPS2b、AGPL1、AGPL2、AGPL3及AGPL4的表达特征。[结果]AGPase 3个同工酶基因AGPS2b、AGPL2和AGPL3表达较强,其中AGPL2相对表达量最高;AGPS2b、AGPL2和AGPL3在2个品种中的相对表达量在适温条件下的均高于高温处理,在特青胚乳中各时期2种温度处理下的相对表达量总体高于泰国香米中的相对表达量。[结论]本研究为进一步利用分子生物学技术培育稳定的优质水稻品种提供理论基础。     

Effects of Dryk1A,ASF and alternative splicing of CaMKⅡδ on myocardial hypertrophy in renovascular hypertensive rats

AIM: To investigate the role of dual-specificity tyrosine phosporylation-regulated kinase 1A (Dyrk1A)-alternative splicing factor (ASF)-calcium/calmodulin-dependent protein kinase Ⅱδ (CaMK Ⅱδ) pathway in the progression of myocardial hypertrophy in renovascular hypertensive rats. METHODS: The renovascular hypertension was induced by two-kidney one-clip (2K1C) method. The changes of blood pressure and myocardial hypertrophy were measured. The techniques of RT-PCR and Western blotting were used to detect CaMKⅡδ alternative splicing and the protein expression of Dyrk1A and ASF, respectively. RESULTS: Eight weeks after operation, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in 2K1C rats increased (P<0.05). The increases in left ventricular weight (LVW), the ratio of LVW to body weight (BW) and the area of myocardial cells indicated that the hypertensive rats developed significant cardiac hypertrophy. The protein expression of Dyrk1A and mRNA expression of CaMKⅡδA and δB were significantly increased, while the protein expression of ASF and mRNA expression of CaMKⅡδC were decreased compared with sham-operated control rats (P<0.05). Treatment with Dryk1A inhibitor epigallocatechin gallate (EGCG) or harmine effectively attenuated cardiac hypertrophy and reversed the changes in the protein expression of Dyrk1A, ASF and alternative splicing of CaMKⅡδ (all P<0.05). CONCLUSION: Dyrk1A-ASF-CaMKⅡδ pathway plays a role in the development of myocardial hypertrophy in renovascular hypertensive rats.     

微小隐孢子虫三个基因主要抗原表位区的串联表达及其抗原性分析

应用多个抗原袁位预测软件对微小隐孢子虫CP15、P23和CP15/60三个子孢子表面抗原的氨基酸序列进行T细胞袁位预测及分析,从中选取了三个抗原表位富集的基因片段,利用重叠延伸PCR(gene splicing by overlapping extension PCR,SOE PCR)将该三个基因片段串联在一起,各基因片段之间以柔性氨基酸(GGGGS)碱基序列链接,得到的拼接片段命名为CpTm.将目的基因克隆到原核表达载体pET-28a(+)上,构建重组表达质粒pET-CpTm,并转化到大肠杆菌BL21(DE3)中进行诱导表达,将纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体.结果成功地构建了CpTm串联基因并在大肠杆菌中以可溶形式高效表达,质谱分析表明重组表达蛋白包含了上述三个抗原的氨基酸序列.Western blot分析显示该重组蛋白能被牛抗微小隐孢子虫阳性血清及隐孢子虫鼠基因型CP15、P23、CP15/60基因重组表达蛋白免疫兔血清识别,制备的抗血清能被重组蛋白特异性识别,表明表达的重组蛋白具有较好的反应原性和免疫原性,为多表位疫苗的研制奠定了基础.     

家蚕乳糖酶-根皮苷水解酶基因BmLPH014192的表达和序列选择性剪接分析

乳糖酶-根皮苷水解酶(lactase-phlorizin hydrolase,LPH)是肠组织上皮细胞微绒膜上的一种糖蛋白,对乳糖和根皮苷有水解活性。鉴定了家蚕基因组中的一个LPH基因,命名为BmLPH014192。该基因CDS长1 305 bp,编码434个氨基酸,预测蛋白质分子质量为50.8 kD,等电点(pI)为5.35。将该基因的cDNA与家蚕基因组序列比对,显示其具有7个外显子,外显子/内含子边界处均符合GT-AG规则。将BmLPH014192与哺乳动物人、褐鼠、家兔和昆虫黑腹果蝇、冈比亚按蚊、二化螟、佛罗里达弓背蚁、大红斑蝶等的LPH进行氨基酸序列比对,相似度都在60%左右;聚类分析中BmLPH014192与昆虫类LPH聚在一起。芯片数据分析结果表明BmLPH014192在家蚕5龄第3天幼虫的9个组织中,只在中肠中有表达,并且表达量很高。在绿茧品种大造和白茧品种19-710幼虫的中肠中扩增到的特异性条带其长度分别为1 000 bp和750 bp,存在序列选择性剪接;在其它白茧品种的中肠中没有扩增出特异性条带。推测BmLPH014192的特殊表达方式可能与其在不同家蚕品种中肠组织中的特异性功能有关。     

SPF大白猪和长白猪CIITA基因剪接突变体的鉴定分析及在不同组织中的表达模式

为了鉴定分析SPF大白猪和长白猪CIITA剪接突变体，研究其在猪不同组织中的表达模式。本研究以5周龄的8头SPF大白猪和7头SPF长白猪为研究对象，设计特异性引物对CIITA基因进行扩增、克隆和测序，对获得的序列分析其剪接突变体特征，利用在线软件对不同剪接突变体进行生物信息学分析，同时利用荧光定量PCR方法检测CIITA基因剪接突变体在2头SPF长白猪8种组织中的表达模式。结果显示，在SPF大白猪和长白猪CIITA基因CDS区存在3种不同的剪接突变体（CIITA-A、CIITA-B和CIITA-C），其CDS长分别为939、984和1 698 bp。CIITA-A和CIITA-B突变体由于Exon 11部分核苷酸缺失（40和199 bp）使终止密码子提前，从而致使编码CIITA蛋白的结构域受到了剪接的影响，缺失了末端富含亮氨酸的4个重复结构域，蛋白结构发生了明显的改变。进化树结果表明，针对CIITA，猪与绵羊和牛具有较近的亲缘关系，在不同猪品种中具有较高的保守性。同时，CIITA不同剪接突变体在猪8种组织中均有表达，且表达量趋于一致，均在脾或肺中表达量较高，其次是肾、脊髓、甲状腺、肝、胸腺，在心脏中表达最低。本研究从SPF大白猪和长白猪上共获得了3个CIITA剪接突变体，其中两个蛋白结构缺失了4个富含亮氨酸的重复结构域，且不同剪接突变体在猪不同组织中均有表达，表达量稍有差异，为后续CIITA基因在机体内的免疫调控功能研究奠定了理论基础。