猪雌激素受体基因研究进展

雌激素受体(ESR)是一种核酸受体，具有转录调控蛋白质的功能，影响着雌激素基因在雌性脊椎动物组织的表达，从而对第二性征、繁殖周期、生殖力、妊娠维持等方面产生影响。本文对雌激素受体的分布、生物学作用、分子结构、基因定位、多态性及其与猪繁殖性能的关系等方面作一综述。     

甘蓝自交不亲和决定因子的体外表达和相互作用的检测

 S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)分别为甘蓝自交不亲和(self-incompatibility, SI)信号传导的雌雄决定因子。为了深入研究两者的作用机理和进行人工调控,本研究以结球甘蓝ZQ为材料,利用pET·NusA融合蛋白表达系统,将包含SRK胞外域和跨膜域的mSRK蛋白和SCR蛋白在大肠杆菌BL21中融合表达,经SDS-PAGE电泳检测表达出融合蛋白大小分别约为116 kD和74 kD。进一步将两融合蛋白进行体外相互作用的检测,结果表明mSRK与SCR蛋白能够相互结合形成稳定的复合体,这为下一步实现SRK-SCR复合体聚合与解离的人为调控提供了技术平台。     

乙烯受体基因LeETR1在番茄Epi和VFN8中的表达及反义表达载体构建

采用RPA方法对番茄乙烯过表达单基因突变体Epi和野生型VFN8中LeETR2 mRNA的表达特征进行了研究。结果表明，在所有被检组织中LeETR2 mRNA均表达，其表达丰度在叶组织中呈发育调节模式，但不受内源乙烯含量的影响；而在果实成熟后期受乙烯的轻微诱导。LeETR2的这种表达模式明显有别于其他乙烯受体基因。为了进一步研究LeETR2的功能，构建了LeETR2反义基因表达载体系统。     

黄牛FSHR基因的生物信息学分析

利用生物基因组学数据库,对黄牛FSHR基因进行生物信息学分析,以预测FSHR基因编码产物的理化性质、结构与功能,同时构建FSHR同源系统进化树.结果表明:FSHR基因编码产物为疏水性跨膜蛋白,具有明显的信号肽,其切割位点位于17～18位的氨基酸之间,二级结构主要以α-螺旋与无规则卷曲为主,并主要在质膜中发挥生物学作用,跨膜区域分为跨膜域、胞外域和胞内域3个部分.序列分析表明,FSHR编码产物可能具有离子通道、运载体、受体、信号转导和阳离子通道等功能,在离子跨膜与信号传递过程中发挥重要作用,并对黄牛的激素调节有影响.FSHR基因编码产物系统进化树表明,黄牛FSHR与水牛、成都麻羊、绵羊等物种FSHR遗传距离较近,具有高度同源性.     

Role of PPARα/PGC-1α in doxorubicin induced mouse dilated cardio-myopathy

AIM: To investigate the changes of peroxisome proliferator-activated receptors (PPAR)α/peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1α) in doxorubicin (DOX) induced dilated cardiomyopathy (DCM) and its effect on the energy metabolism and myocardial function in mice. METHODS: Forty mice were randomly divided into 4 groups: control group, DOX group, PPARα inhibitor group and PPARα agonist group. The DCM model was established by injection of DOX. The protein levels of PPARα/PGC-1α were detected. The PPARα inhibitor and PPARα agonist were used 2 weeks beforeinjection of DOX. The contents of adenine acid and phosphocreatine (Pcr) in the mitochondria were measured by high-performance liquid chromatography (HPLC). The ANT activity was analyzed by the atractyloside-inhibitor stop technique. The changes of the echocardiography and hemodynamics were also observed. RESULTS: DOX induced DCM model was successfully established. The protein levels of PPARα and PGC-1α in control group were significantly higher than those in DOX group (P<0.05). Both of the high-energy phosphate contents and the transport activity of ANT were decreased in DOX group (P<0.05), and the hemodynamic parameters were disordered (P<0.01). Compared with DOX group, PPARα inhibitor pre-treatment significantly reduced the PPARα/PGC-1α expression. Meanwhile, high-energy phosphate contents in the mitochondria and the ANT transport activity of the mitochondria decreased, as well as the left ventricular function (P<0.05). On the other hand, PPARα agonist significantly increased the expression of PPARα and PGC-1α, and improved the transport activity of ANT. In addition, the hemodynamic parameters were ameliorated, but the high-energy phosphate contents of the mitochondria did not significantly change. CONCLUSION: PPARα/PGC-1α plays an important role in the regulation of ANT transport activity in dilated cardiomyopathy induced by DOX, and the activation of PPARα/PGC-1α has protective effects on the DCM induced by DOX.     

Corticosterone inhibits LPS-induced expression of NLRP3 in mouse macrophages and its relation with XO

AIM: To investigate the inhibitory effect of corticosterone (CORT) on lipopolysaccharide (LPS)-induced expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and its relation with xanthine oxidase (XO). METHODS: An inflammatory model of mouse macrophage RAW 264.7 was established by stimulating with LPS. Total cellular protein was extracted after the macrophages were treated with CORT at different concentrations (0~900 μg/L). The protein levels of NLRP3 and caspase-1 were determined by Western blot. According to the treatments, the macrophages were divided into control group, LPS group, LPS+CORT group and LPS+allopurinol group. Cell components were extracted at 0, 0.5, 1, 1.5 and 2 h. The protein levels of NLRP3 and XO were determined by Western blot,and the mRNA expression of NLRP3 and XO was detected by real-time PCR. RESULTS: CORT at 700 μg/L and above significantly inhibited the expression of NLRP3 and the activation of caspase-1 in the macrophages induced by LPS (P<0.05). Compared with LPS group, the expression of NLRP3 and XO in LPS+CORT group was inhibited (P<0.05), and the expression of NLRP3 in LPS+allopurinol group was also reduced (P<0.05).CONCLUSION: High concentration of CORT inhibits the expression of NLRP3 in LPS-induced mouse macrophages, which is associated with XO. The inhibitory effect of CORT may be related to the reduction of XO expression.     

A combined application of molecular docking technology and indirect ELISA for the serodiagnosis of bovine tuberculosis

BackgroundThere is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity.ObjectivesTo develop a reliable and rapid strategy to improve diagnostic tools for bTB.MethodsIn this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect.ResultsThe results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen.ConclusionsMolecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.     

荷斯坦乳牛乳腺和乳腺上皮细胞中Toll样受体及辅助因子编码基因的检测

参照牛TLR4、TLR2、CD14、MD-2基因序列设计了相应基因的引物。采用RT-PCR技术检测了体外培养的荷斯坦乳牛乳腺和乳腺上皮细胞中Toll样受体TLR4、TLR2及辅助因子CD14、MD-2基因。结果显示,乳腺上皮细胞中存在TLR4、TLR2、CD14和MD-2四个基因的表达,而乳腺中除MD-2未检测到外,其余3个基因均扩增成功。说明该受体及辅助因子可能参与了乳腺的先天性免疫防御。该研究为探讨乳腺的先天性免疫及乳腺上皮细胞在乳腺先天性免疫中的作用奠定了基础。     

Adenovirus-mediated mPPARγ1 gene overexpression inhibits IFN-γ-induced galectin-9 gene and protein expression in ECV304

AIM:To investigate whether adenovirus-mediated mPPARγ1 gene overexpression inhibits IFN-γ-induced galectin-9 gene and protein expression in ECV304. METHODS:A replication-deficient recombinant adenovirus expression vector of mPPARγ1 was constructed by using the AdEasy system. ECV304 were incubated for 24 h with 1×104 U/L, 5×104 U/L, 1×105 U/L and 2×105 U/L IFN-γ, respectively. ECV304 stimulated with 1×105 U/L IFN-γ were divided into 4 groups in random: P group (PPARγ1 gene overexpression), T group (treated with troglitazone 40 μmol/L in DMSO), PT group (PPARγ1 gene overexpression+troglitazone treatment) and control group. Changes of PPARγ and galectin-9 in mRNA and protein levels in different groups and subgroups were investigated by RT-PCR and immunoblotting. RESULTS: Galectin-9 expression was very few in normal ECV304. IFN-γ induced the expression of galectin-9 in ECV304. Degree of galectin-9 expression increased with the dose of IFN-γ. PPARγ1 gene overexpression inhibited IFN-γ-induced galectin-9 expression in ECV304. Galectin-9 mRNA and protein expressions from PT group and P group were inhibited in similar degree (P>0.05). However, this effect was not observed in troglitazone intervention (P>0.05). PPARγ expression was also very few in normal ECV304. PPARγ1 gene overexpression/activation had no effect on endogenous mPPARγ expression. CONCLUSION: This may partly contributed to the anti-inflammatory and immuno-regulatory effect of PPARγ1 gene overexpression by inhibiting IFN-γ-induced galectin-9 gene and protein expression in ECV304.