3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

大肠杆菌整体代谢网络调控基因(csrA)的敲除及其对苯丙氨酸生物合成的影响

csrA基因产物是大肠杆菌芳香族氨基酸生物合成途径中碳中心代谢有关的一种全局性调控蛋白质。采用Red敲除系统介导的同源重组的方法定位缺失大肠杆菌染色体csrA基因,经PCR、DNA测序等多种方法证实了基因重组缺失的可靠性。csrA基因缺失后,缺失菌株较对照菌株,糖酸转化率有所提高,发酵生产苯丙氨酸的能力也得到一定的提高,产酸提高约13%。     

口蹄疫病毒3A基因在大肠杆菌中的高效表达

将口蹄疫病毒(Foot\|and\|Mouth Disease Virus,FMDV)的3A基因克隆到线形化的原核表达载体pProEX\|HTb中,转化大肠杆菌BL21和DH5α,经氨苄抗性筛选得到阳性克隆,IPTG诱导表达。SDSPAGE和West bolt结果证实大肠杆菌菌体不可溶性蛋白中富含3A蛋白,说明3A蛋白在表达产物中以包涵体的形式存在,所表达的蛋白含量占菌体蛋白的29.2%。     

Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus

The four tropane alkaloids have played a pivotal role in controlling diseases such as the toxic and septic shock, the organophosphorus poison and the acute lung injury. Here, the elicitation effect of different elicitors on the production of tropane alkaloids and the molecular mechanism of enzyme genes in the pathway was firstly demonstrated in hairy roots of Anisodus acutangulus. The results showed ethanol, methyl jasmonate and Ag⁺ could improve the accumulation of tropane alkaloids up to 1.51, 1.13 and 1.08 times after 24 h treatment, respectively (P < 0.05), whereas salicylic acid decreased the average content of tropane alkaloids. Furthermore, expression profile analysis results revealed that up-regulation of hyoscyamine-6b-hydroxylase (AaH6H) and little regulation of tropinone reducase II (AaTR2) elicited by ethanol, increased expression of putrescine N-methyltransferase I (AaPMT1) elicited by Ag⁺, elevated expression of tropinone reducase I (AaTR1) elicited by methyl jasmonate, respectively, resulted in tropane alkaloids improvement. Our results showed that hairy root culture of A. acutangulus in combination with elicitors was a promising way for production of tropane alkaloids in the future.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

硝酸镧浸种对NaCl胁迫下柳枝稷种子萌发及幼苗生理特性的影响

为探讨镧对NaCl胁迫下柳枝稷种子萌发及幼苗生理特性的影响,该研究以柳枝稷(Panicum virgatum L.)种子为研究材料,通过不同浓度(0、0.05、0.1、0.5和1.0mmol/L)La(NO3)3溶液浸种24h处理后,添加100mmol/L NaCl溶液,以蒸馏水为对照,在种子发芽箱中培养并观察柳枝稷种子萌发和幼苗生长,测定幼苗叶片相对电导率、丙二醛、脯氨酸和叶绿素的含量。结果显示:(1)0.05mmol/L La(NO3)3浸种处理可缓解100mmol/L NaCl对种子发芽的影响。(2)不同处理对幼苗苗长无显著影响,0.1mmol/L La(NO3)3浸种显著增加了幼苗的鲜重和叶面积,当浓度≥0.5mmol/L时根长、根冠比、鲜重和叶面积受到抑制。(3)幼苗叶片相对电导率和脯氨酸含量随La(NO3)3浓度的增加呈先降低后增加的趋势,其中0.05和0.1mmol/L La(NO3)3浸种处理下效果较好,丙二醛含量随La(NO3)3浓度的增加持续降低,但各浓度处理间差异不显著,叶绿素含量在0.05mmol/L La(NO3)3浸种处理下较NaCl处理显著增加,当浓度≥0.5mmol/L随着浸种浓度的增加呈下降趋势。(4)不同指标间发芽势与发芽率、根长和叶绿素含量间呈极显著正相关关系,与根冠比、电导率和脯氨酸含量均呈极显著负相关关系,而与苗长不相关。研究表明,低浓度(0.05mmol/L)La(NO3)3可缓解NaCl胁迫对柳枝稷种子萌发及幼苗生长的影响,而高浓度(1.0mmol/L)的La(NO3)3则会加重NaCl的胁迫危害。     

圈养小熊猫繁殖行为变化及繁殖行为对策

In order to investigate the change of reproductive behaviors and understand reproductive strategies of both male and female red pandas, one-year behavioral observation was conducted through the focal sampling method in the Chengdu Research Base of Giant Panda Breeding from December 1999 to November 2000. Our results indicated that reproductive behaviors showed significant differences between the estrous and non-estrous seasons. Frequencies of the rubbing anogenital, sniffing and licking marking were much higher in estrus than in non-estrus. Bleating only appeared in the estrus and can be regarded as an estrous indicator.The result also demonstrated that both male and females applied different reproductive behavioral strategies. Frequencies of activity, rubbing anogenital, licking and sniffing marking, and bleat were much higher in the male than in the female. However, those of resting and investigating were much lower in the male than in the female. This indicated that the male was more active than the female during the estrus and might imply that the male acts mainly as an estrous message sender, and the female as a message receiver in the estrous season.     

Structural basis for the specific recognition of RET by the Dok1 phosphotyrosine binding domain

Dok1 is a common substrate of activated protein-tyrosine kinases. It is rapidly tyrosine-phosphorylated in response to receptor tyrosine activation and interacts with ras GTPase-activating protein and Nck, leading to inhibition of ras signaling pathway activation and the c-Jun N-terminal kinase (JNK) and c-Jun activation, respectively. In chronic myelogenous leukemia cells, it has shown constitutive phosphorylation. The N-terminal phosphotyrosine binding (PTB) domain of Dok1 can recognize and bind specifically to phosphotyrosine-containing motifs of receptors. Here we report the crystal structure of the Dok1 PTB domain alone and in complex with a phosphopeptide derived from RET receptor tyrosine kinase. The structure consists of a beta-sandwich composed of two nearly orthogonal, 7-stranded, antiparallel beta-sheets, and it is capped at one side by a C-terminal alpha-helix. The RET phosphopeptide binds to Dok1 via a surface groove formed between strand beta5 and the C-terminal alpha-helix of the PTB domain. The structures reveal the molecular basis for the specific recognition of RET by the Dok1 PTB domain. We also show that Dok1 does not recognize peptide sequences from TrkA and IL-4, which are recognized by Shc and IRS1, respectively.     

Amphibian tachykinin precursor

The precursor of amphibian tachykinin has not been found although more than 30 tachykinins have been isolated from amphibians since 1964. In this report, two tachykinin-like peptides are identified from the skin secretions of the frog, Odorrana grahami. Their amino acid sequences are DDTEDLANKFIGLM-NH(2) (named tachykinin OG1) and DDASDRAKKFYGLM-NH(2) that is the same with ranamargarin found in Rana margaretae, respectively, with a conserved FXGLM-NH(2) C-terminal consensus motif. By cDNA cloning, their precursors were screened from the skin cDNA library of O. grahami. The precursors are composed of 61 amino acid (aa) residues including a signal peptide followed by an acidic spacer peptide and one copy of mature tachykinin-like peptide. Their overall structure is different from structures of other tachykinin precursors such as human protachykinin 1 precursor containing 143 aa including one copy of substance P (SP) and neurokinin A (NKA), and ascidian tachykinin 1 precursor containing 164 aa including two copies of tachykinin-like peptides. The current results demonstrate that the biosynthesis mode of tachykinins in amphibians is different from other animals.     

Purification and characterization of two new allergens from the venom of Vespa magnifica

Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.