Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.     

Preparation of m1AChR-G11 fusion protein and m4AChR-G16 fusion protein and effects of various ligands on them

AIM:To prepare m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein in Baculovirus-Sf9 cell system and detect the effects of various muscarinic ligands on the interaction between m₁AChR and G₁₁ and m₄AChR and G₁₆, and screen different kinds of ligands specific for m₁ and m₄. METHODS:To prepare fused DNA of m₁AChR-G₁₁and m₄AChR-G₁₆ in two PCR, then expressed in Sf9 cells and detect the pharmacological function of m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein by QNB and GTPγS binding experiments; To expore the way of the activation of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein by various ligands includingcetylcholine (ACh), Pilocarpine (Pilo), 4-hydroxy-2-butynyl-1-trimethylammonium-m-chloro-carbanilatechloride (McN-A-343), tetrandrine, pirenzepine (PZ), alcuronium, atropine, R-(+)-hyoscyamine and gallamine by displacement by GDP on GTPγS binding experiments. RESULTS:The expression levels of m₁AChR-G₁₁ and m₄AChR-G₁₆ fusion protein were (45.39±2.62) nmol·g^-1 protein, (47.04±1.58) nmol·g^-1 protein. The affinity of GDP to G₁₁ and G₁₆ partner changed in the presence of different muscarinic ligands. CONCLUSION: The m₁AChR-G₁₁ and m₄AChR-G16 showed the pharmacological specificity to m₁ and m₄ receptor and the efficient signaling of the two partners. Ligands of m₁AChR and m₄AchR mediated different signal transduction by changing the affinity of G₁₁/G₁₆ and GDP. So m₁AChR-G₁₁ fusion protein and m₄AChR-G₁₆ fusion protein can be taken as a tool to screen ligands specific for m₁AChR and m₄AChR.     

Pharmacological analyses of two naturally occurring porcine melanocortin-4 receptor mutations in domestic pigs

The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Numerous mutations in the MC4R gene have been identified from obese humans. So far two naturally occurring porcine MC4R (pMC4R) mutations, D298N and R236H, have been identified from various strains of pigs and D298N is being utilized as a genetic marker to screen performance traits of pigs. In this study, we performed functional analyses of pMC4R D298N and R236H, including their ligand binding and signaling properties in transiently transfected HEK293T cells. Ligand binding assays showed that both D298N and R236H pMC4Rs had similar binding capacities and affinities for the natural agonist -MSH and the natural antagonist Agouti-related protein as wild-type pMC4R. In signaling assays, both mutants had normal EC₅₀ and maximal signaling to -MSH. In summary, pMC4R mutants D298N and R236H do not have any overt functional defects; therefore we suggest caution using these mutations as selection markers in breeding programs.     

美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析

通过比对分析已知昆虫氨肽酶N(aminopeptidase N,APN)氨基酸序列并结合本实验室的美国白蛾(Hyphantria cunea)中肠iTRAQ结果,设计了hcapn3基因特异引物,获得hcapn3基因片段。通过RACE-PCR技术获得美国白蛾中肠氨肽酶N基因(hcapn3)全长序列(GenBank登录号为KJ013598)。Blastp分析表明,获得的氨肽酶属于氨肽酶N3家族,命名为HcAPN3。序列分析显示HcAPN3包括952个氨基酸残基,具有典型的谷氨酸锌化氨肽酶(Gluzincin)结构域和羧基端(ERAP1_C)结构域。利用Bac to Bac表达系统在昆虫细胞中表达108kDa的HcAPN3蛋白。在原核表达系统中表达58kDa的Gluzincin结构域和49kDa ERAP1_C的结构域蛋白。Ligand Blot分析结果显示,HcAPN3蛋白及Gluzincin结构域可与Cry1Ac蛋白特异性结合,但ERAP1_C结构域未能与Cry1Ac结合。本研究首次克隆了美国白蛾氨肽酶基因并分析了HcAPN3与Cry1Ac的结合特性,为下一步功能研究提供基础。     

美国白蛾氨肽酶N的基因克隆表达及与苏云金杆菌3种毒素蛋白的结合特性分析

美国白蛾(Hyphantria cunea)是桑树的重要害虫。为明确苏云金杆菌(Bacillus thuringiensis)杀虫晶体蛋白对该虫的作用机制,利用已知昆虫氨肽酶N基因序列设计引物,以美国白蛾中肠cDNA为模板,利用RACE-PCR技术获得美国白蛾中肠氨肽酶N基因hcapn1全长序列(GenBank登录号:KP053647)。该基因开放阅读框为3 000 bp,编码999个氨基酸,预测蛋白质的分子质量和等电点分别为113.14 kD和4.85。设计去信号肽引物PCR扩增获得hcapn1基因序列,构建原核重组表达载体pET30a-hcapn1,诱导表达的HcAPN1重组蛋白约110 kD。将苏云金杆菌Cry1Ac、Cry2Ab33和Cry9Ea6原毒素经胰蛋白酶消化后与HcAPN1重组蛋白分别进行体外配体印迹试验,发现HcAPN1重组蛋白与Cry9Ea6结合,而不与Cry2Ab33、Cry1Ac发生特异结合,推测HcAPN1可能为Cry9Ea6在美国白蛾中肠的受体蛋白。分别设计引物扩增HcAPN1蛋白2个结构域Asp₃₄～Asp_( 527)和Leu₅₆₃～Gln₉₂₅的编码序列,在大肠埃希菌中表达获得62 kD和48 kD 2种重组蛋白,体外结合试验显示Cry9Ea6与HcAPN1的结合发生在Leu₅₆₃～Gln₉₂₅之间。研究结果为深入理解苏云金杆菌Cry9Ea6蛋白对美国白蛾的杀虫机制提供了基础数据。     

醋酸——醋酸铵缓冲体系中柠檬酸对高岭石的溶解特征

采用间歇法(batch method)模拟研究醋酸-醋酸铵缓冲体系中柠檬酸对高岭石的溶解特征。结果表明:反应液中Al、Si浓度随柠檬酸浓度提高、反应液pH降低及反应时间的推移而增加;且低浓度柠檬酸(1mmol L-1)时,随着酸度的升高,配体促进溶解速率(RL)减小;高浓度柠檬酸(≥5mmol L-1)时,随着酸度的升高,配体促进溶解速率(RL)增加,但增幅随pH的降低而大幅减弱;且随着酸度的升高,配体对高岭石溶解的贡献相对减弱,但相对于质子,本试验柠檬酸浓度及酸度范围内,其对高岭石溶解的贡献仍是主要的。     

亲和层析纯化猪胰蛋白酶的研究

采用猪胰蛋白酶的天然抑制剂-鸡卵类粘蛋白(CHOM)作为配基合成亲和吸附剂,从猪胰脏的粗提液中,通过亲和层析直接获得高纯度的胰蛋白酶.此法具有操作简便、产品纯度、比活力和获得率高等优点.     

Selective ligand-binding determinants in catfish and human gonadotropin receptors

In mammals, the specificity of FSH–FSH receptor (FSHR), LH–LH receptor (LHR) and TSH–TSH receptor couples is such that no cross-activation occurs under normal physiological conditions. The interactions between fish gonadotropins and their receptors, however, appear to be less discriminatory. For example, the catfish FSHR is highly responsive to both catfish LH and catfish FSH, while the catfish LHR is specific for its cognate LH. Comparative structure–function studies aimed at elucidating the molecular basis of ligand promiscuity (in fish) and ligand selectivity (in mammals) are described in this paper.     

核受体因子PPARγ的研究进展

过氧化物酶体增殖剂激活受体γ(PPARγ)是核激素受体超家族成员.PPARγ具有多种生物学效应,对机体起重要作用,是目前的研究热点.本文对PPARγ的结构、作用机理以及生物学作用等进行了综述.     

磷酸根在矿物表面的吸附-解吸特性研究进展

综述了磷酸根在一些常见土壤矿物表面吸附-解吸特性的研究进展.磷酸根在矿物表面的吸附特性受环境pH、离子强度、温度、反应时间、矿物类型等多种因素的共同影响.一般说来,矿物表面的磷吸附量随pH降低而增加,受离子强度的影响较小.磷酸根在矿物表面的吸附动力学过程可分为快速吸附过程和慢速吸附过程,且在弱结晶矿物中存在微孔扩散过程...