The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene

Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene, which is difficult to package in the promising adeno-associated virus (AAV) vectors. Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain. Previously, we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene, suggesting that the light chain, covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII. However, its level of secretion was affected by inefficient secretion the heavy chain. Here, we studied the effect of a mutant heavy chain with L303E/F309S substitutions, which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene. Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells. A spliced FVIII protein was seen in co-transfected cells by Western blot analysis. The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene, which was greater than the secretion of wild-type heavy chain. The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL, with an activity of (0.61±0.08) IU/mL, which was greater than that of wild-type FVIII co-transfected cells. Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections. This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector. These results encourage the transfer of this technology to an animal model using a dual-AAV vector.     

大果水晶梨与其褐色突变体多酚氧化酶及过氧化物酶同工酶差异(简报)

大果水晶梨果皮为绿色,2007年在其植株上发现一果实呈褐色的变异枝,经2008-2009年嫁接试验后确定为遗传性变异。与原品种比较,褐色突变体果实多了一层褐色木栓层。     

A spontaneous benomyl-resistant mutant ofPodospora anserina exhibiting a diurnal growth rhythm

Summary A spontaneous mutant ofP. anserina isolated by screening for benomyl resistance exhibited a diurnal growth rhythm dependent on light-dark cycles. The rhythmic character, the benomyl resistance and a growth rate reduced to 50% of that of the wild type were inherited together over more than 10 generations. The locus was mapped on linkage group II, 0.35 map units distal to the locusz (=0.81 map units from the centromere).     

水稻细菌性条斑病菌一个假定的udgH基因的功能研究

水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)是一种重要植物病原菌,其引发的水稻细菌性条斑病导致水稻严重减产。Xoc一个假定的udgH基因的突变,导致Xoc产胞外多糖和致病力的严重减弱,并使胞外淀粉酶和纤维素酶的酶活也有一定的降低,互补菌株能恢复到野生型水平,但致病力仅能恢复到80%,而高表达菌株与野生型相比胞外多糖产量增加,但致病力降低。本研究为进一步研究该基因在水稻条斑病菌中胞外多糖代谢途径的作用奠定了基础,并为利用基因工程手段改造高产黄原胶菌种提供了一种可行的策略。     

虎纹捕鸟蛛毒素-IV突变体K27A-HWTX-IV的合成和复性及其生物学活性研究

用芴甲氧羰基(Fmoc)固相多肽合成的方法在固相多肽合成仪上合成了用丙氨酸代替虎纹捕鸟蛛毒素—Ⅳ的第27位的赖氨酸的突变体K27A—HWTX—Ⅳ，通过反相HPLC对不同条件下突变体的氧化复性结果进行监测，摸索出其最佳氧化复性条件为：0．1mol／L Tris—HCl，pH8．0，GSH 1 mmol／L GSSG 0．1mmol／L的复性缓冲液，样品浓度为0．1mg/mL。复性产物经HPLC纯化并用基质辅助激光解吸飞行时间质谱法进行鉴定，相对分子质量为4050．63Da。生物学活性实验小鼠离体膈神经—膈肌标本测活实验表明：10μg/mL天然HWTX—Ⅳ阻断小鼠离体膈神经,膈肌接头传递时间为16min，10μg/mL复性后的突变体阻断小鼠离体膈神经．膈肌接头传递时间为120min，残基的替换导致突变体生物学活性比天然毒素少，证明了第27位的赖氨酸为虎纹捕鸟蛛毒素—Ⅳ活性相关的残基。     

无毛突变基因对无毛小鼠免疫器官结构及功能的影响

摘要: 目的探讨无毛突变基因的作用,对无毛小鼠、有毛小鼠的免疫器官结构及功能进行了对比研究。方法比 较无毛小鼠和有毛小鼠主要免疫器官组织学变化,以及血清白介素－ 2 受体、淋巴细胞亚群、淋巴细胞增殖等方面 的差别。结果发现无毛小鼠和有毛小鼠免疫器官结构及功能均有一定的差异。结论研究结果提示无毛突变 基因不仅影响被毛结构,对免疫器官及功能也有一定的影响。该基因在杂合状态时也有一定的作用,表明该突变 基因具有一定的共显性。     

污泥在人工鱼礁制备中的试验研究

为了有效地利用污泥以及促进人工鱼礁的发展,以污泥、水泥、贝壳粉为原料按照不同的水灰比和贝壳粉含量制作了人工鱼礁礁块并对其进行抗压强度、营养盐析出以及浸泡海水液pH的测试,同时用显微镜和X射线衍射对礁块水化产物进行微观分析。试验结果表明:在水灰比为0.5～0.6、贝壳粉含量为0.2～0.6的范围内,水灰比越低或贝壳粉含量越高均能使礁块的强度更高;礁块掺加污泥后可以不断析出NH~+_4和PO~(3-)_4且析出规律均为线性,但当材料比例改变时这种线性之间也会有差异;强度越高的礁块其浸泡海水液的pH越接近普通海水,亲水性越好;礁块水化生成钙矾石和C-S-H凝胶等是礁块强度的重要保障。该研究可为污泥的资源化利用以及人工鱼礁材料的新型开发提供思路和技术参考。     

Cloning and functional analysis of the sequences flanking mini-Tn5 in the magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1

A magnetosome-deleted mutant NM21 of Magnetospirillum gryphiswaldense MSR-1 was generated by mini-Tn5 lacZ2 transposon mutagenesis, and a 3073-bp fragment flanking mini-Tn5 lacZ2 in NM21 was cloned by Anchored PCR. Sequencing analysis showed that this fragment involved three putative ORFs; the mini-Tn5 lacZ2 was inserted into ORF1. Functional complementary test indicated that the 3073-bp fragment was required for biosynthesis of magnetosomes in M. gryphiswaldense MSR-1. The majority of proteins, which had homology with the protein encoded by ORF1, were the cation transporter. Transmembrane domain analysis showed that the protein encoded by ORF1 contained four transmembrane domains. It may be a transmembrane protein. The protein encoded by ORF1 contained two putative conserved domains: COG0053 and PRK09509. The MMT1 and FieF, containing conserved domains COG0053 and PRK09509 too, were Fe²⁺ transporter (cation diffusion facilitator superfamily). It was suggested that the protein encoded by ORF1 might take part in the magnetosomes biosynthesis as Fe²⁺ transporter. Supported by National Natural Science Foundation of China (Grant No. 30570023) and Scientific Research Project of Huaibei City, Anhui Province (Grant No. 070114)     

白桦转BpGH3.5基因叶片早衰突变株的光合特性及生长分析

【目的】植物叶片早衰将影响作物产量和品质,研究叶片早衰机制对于培育耐早衰优良品种具有重要意义。【方法】以转BpGH3.5基因的白桦叶片早衰突变株(G4)、非转基因白桦(WT)及叶片正常的转基因白桦(G21)等为材料,测定其叶绿素含量、净光合速率(P_n)、气孔导度(G_s)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)等光合指标,测定苗高的时序变化规律。【结果】叶片早衰突变影响了叶片叶绿素的合成及积累,突变株的叶绿素相对含量(SPAD)均值为36.08,相对两个对照株系分别下降7.34%、7.48%。叶片早衰突变影响了白桦光合呼吸作用。突变株的净光合速率、气孔导度、蒸腾速率分别为WT野生株系的67.54%、64.44%、64.93%,胞间CO₂浓度达到234.33 μmol/mol,显著高于G21对照株系( P<0.05)。突变株的当年高生长显著低于WT、G21 两个对照株系,当年高生长分别是WT、G21 两个对照株系的68.9%、85.0%。利用Logistic方程对3个参试株系当年苗高生长量的变化过程进行了拟合,其系数均高于0.98,同时,通过Logistic方程计算的生长参数揭示了早衰突变株高生长较两个对照株系低的原因是速生期苗高平均生长量(GR)、苗高日生长量的平均值(GD)等生长参数较低。【结论】转BpGH3.5基因的白桦发生了叶片早衰现象,使叶绿素提前降解,影响了光合呼吸作用,进而影响了苗高生长。