基于乌金猪深度发掘的优质高效新品种选育

目的揭示宣和猪PRKAG3基因外显子5多态性及其与肉质性状的关联。方法采用PCR产物直接测序法检测了120头宣和猪PRKAG3基因外显子5区域的SNP位点,借助最小二乘模型分析了各位点不同基因型及合并基因型对6个肉质性状的影响。结果宣和猪PRKAG3基因外显子5区域检出3个SNP位点,包括2个同义突变位点(T579C、T580C)和1个错义突变位点(G595A),分别以TT、TT、GA基因型和T、T、G等位基因的频率最高,且都处于Hardy-Weinberg平衡状态。T579C、T580C位点的TT和TC基因型可显著提高大理石纹(P<0.05)和降低失水率(P<0.01),分别呈显著的加性效应及加性—显性效应(P<0.05或P<0.01);G595A位点的AA基因型失水率最低(P<0.05)、熟肉率最高(P<0.05),呈显著的加性效应(P<0.05);3个位点的合并基因型TTTTGG、TCTCGG和TTTTAA分别具有最高的大理石纹、pH₁和最低的失水率(P<0.05或P<0.01),CCCCGG基因型的大理石纹和pH₁最低、失水率最高(P<0.05或P<0.01)。结论研究结果进一步确认宣和猪PRKAG3基因外显子5多态性与肌肉大理石纹、pH值和失水率显著关联,T579C、T580C位点的CC基因型和G595A位点的GG基因型具有协同降低猪肉品质的效应。     

Influence of paricalcitol on renal tubulointerstitial fibrosis in diabetic nephropathy

AIM: To investigate the effect of paricalcitol (P) on renal tubulointerstitial fibrosis and the underlying mechanisms in diabetic nephropathy (DN).METHODS: DN rat model was induced by a single intraperitoneal injection of streptozotocin after fasting. The animals were randomly divided into 2 groups:the DN rats in paricalcitol-intervened group (group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4 μg/kg (3 times a week); the DN rats in DN group (group D) were given isopyknic propylene glycol. Normal control group (group C) was also set up. The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks. The biochemical indexes were measured. The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4, β-catenin and Klotho by immunohistochemistry and Western blotting. In addition, the correlation among the above indexes was analyzed.RESULTS: (1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D (P<0.05). (2) The area of renal tubulointerstitial fibrosis increased in group D compared with group C, while decreased in group P compared with group D (P<0.05). (3) The expression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05). Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 and β-catenin decreased in group P (P<0.05). (4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 and β-catenin (P<0.05).CONCLUSION: Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.     

Effects of baicalin on glial fibrillary acidic protein and nuclear factor-κB expression in juvenile rat hippocampus after status convulsion

AIM: To study the effects of baicalin (BC) on glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) expression and neuronal apoptosis in juvenile rat hippocampus after status convulsion (SC). METHODS: One hundred and ninety five juvenile male Sprague-Dawley rats were randomly divided into 3 groups: normal saline pretreatment group (NS group), SC group and SC with BC pretreatment group (BC group). Each of these 3 groups would be subdivided into 5 subgroups sacrificed at 4 h, 12 h, 24 h, 48 h and 72 h after SC. The rat SC model was prepared by lithium-pilocarpine chemical method. The protein expression of GFAP and NF-κB was detected by the method of immunohistochemistry. The mRNA expression of GFAP was detected by RT-PCR. The neuronal apoptosis was observed by TdT-mediated dUTP nick end labeling (TUNEL). RESULTS: Compared with NS group, the GFAP positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of GFAP was significantly reduced in BC group (P<0.05). Compared with NS group, the NF-κB positive cells was increased in SC group (P<0.05). Compared with SC group, the expression of NF-κB was significantly reduced in BC group. RT-PCR showed that the expression trend of GFAP mRNA was similar to that of the protein. Compared with NS group, the TUNEL positive cells in the hippocampus CA1 area in SC group increased significantly 12 h after SC (P<0.01), and reached a peak at 48 h. After the intervention with BC, the TUNEL positive cells decreased significantly between 12~48 h after SC (P<0.05 or P<0.01), but the number of TUNEL positive cells remained significantly greater than that in NS group (P<0.05). CONCLUSION: The expression of GFAP and NF-κB in the hippocampus increased after SC in rats. Baicalin decreases the expression of GFAP and NF-κB in hippocampus of rats with pilocarpine-induced seizures, and reduces the number of neuronal apoptosis, suggesting that baicalin may protect against the brain damage caused by status convulsion.     

骆驼乳清蛋白对热应激所致大鼠肝氧化损伤的保护作用

本试验旨在研究骆驼乳清蛋白（CWP）对热应激（HS）大鼠肝损伤、氧化应激及肝功能的保护作用。选取6周龄SD大鼠36只,适应性饲养2周后随机分为6组：正常对照组饲喂基础日粮;CWP对照组添加400 mg·kg^-1的CWP;HS组除饲喂基础日粮外,每天进行2 h HS处理,连续8 d;3个CWP干预组分别于每次HS前灌服100、200和400 mg·kg^-1的CWP。试验结束后,取大鼠肝组织,HE染色观察肝组织病理学变化,同时检测肝功能生物标志物、氧化应激标志物水平及抗氧化酶活性。结果表明：CWP降低了HS大鼠血清谷草转氨酶（AST）和谷丙转氨酶（ALT）活性,400 mg·kg^-1的CWP干预效果最好（P<0.01）;400 mg·kg^-1CWP干预组有效降低了HS诱导的大鼠肝组织病理学改变; CWP降低了大鼠肝活性氧（ROS）及丙二醛（MDA）水平,增加了超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活性及谷胱甘肽（GSH）水平,400 mg·kg^-1的CWP干预效果最好（P<0.01）。结果提示,CWP干预能够以剂量依赖性方式降低大鼠肝氧化应激,增加抗氧化系统防御能力,从而缓解HS所致大鼠肝损伤。     

干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1,IFITM1）对猪源冠状病毒复制的影响,本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后,实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes,ISGs）的mRNA表达水平;利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长,分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中,分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒,慢病毒侵染PK15细胞后用嘌呤霉素进行筛选,获得稳定表达及稳定干扰IFITM1表达的PK15细胞系,分别命名为PK15-IFITM1及PK15-IFITM1^-/-,并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况;TGEV接种PK15-IFITM1^-/-和PK15-IFITM1,实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示,PK15细胞接种TGEV后的48 h内,一些ISGs的mRNA水平均有所上升;PK15-IFITM1^-/-细胞系的干扰效率为70%,PK15-IFITM1细胞系表达成功;在PK15-IFITM1^-/-细胞系中,IFITM1的mRNA水平显著下调,促进了TGEV的复制。反之,在PK15-IFITM1细胞系中,TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之,IFITM1对TGEV有显著的抗病毒作用,IFITM1不影响TGEV对PK15细胞的早期吸附,这为后续IFITM1抗冠状病毒机制的研究奠定了基础。     

The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP⁺ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.     

Effect of Se and VE Supplement on Semen Quality,Antioxidant Enzyme Activities and Heat Shock Protein Expression of Goat in High Temperature Season

This experiment was conducted to study the effect of selenium and vitamin E supplement on semen quality, antioxidant enzyme activities and heat shock protein expression of goat in Hainan high temperature season.16 adult Hainan Black goat with good health and approximate weight were randomly divided into 4 groups, fed with basal diet(control group), basal diet+0.5 mg/kg Se(Se group), basal diet+100 mg/kg VE(VE group), and basal diet+0.5 mg/kg Se+100 mg/kg VE(Se+VE group), respectively.The experimental period was 93 d.Semen samples were collected in the last week of the experiment on two consecutive days.The semen quality, antioxidant enzyme activities and heat shock protein expression were analyzed.The results showed that compared with control group, the ejaculate volume was not significantly affected by Se or VE supplement(P>0.05).Sperm density and sperm motility were increased significantly by Se and VE supplement(P<0.05), and the abnormal rate was decreased extremely significantly(P<0.01).The goats fed with Se and VE also had higher activities of GSH-Px(P<0.01), SOD(P<0.05), CAT(P<0.05) and T-AOC(P<0.01), and lower MDA concentration in seminal plasma(P<0.05).The relative expression levels of HSP70 and HSP90 mRNA in supplement groups were decreased extremely significantly(P<0.01).However, there were some certain differences between the Se and VE supplement groups on semen quality and heat shock protein expression.In conclusion, the supplementation of Se and VE could help to improve goat semen quality by increasing the sperm density, sperm motility, the antioxidant enzyme activities, and decreasing the abnormal rate in hot season of Hainan.Finally, Se and VE supplement had good effects on relieving the environment heat stress.     

Effects of Dietary Crude Protein Levels on Growth Performance,Digestion and Metabolism,and Serum Biochemical Indexes of Super Merino Lamb

The experiment aimed to find out the effects of dietary crude protein levels on growth performance, digestion and metabolism and serum biochemical indexes of Super Merino lamb after weaning.According to the dietary crude protein level, 64 Super Merino weaned lambs were divided into four groups, which the crude protein levels were 13.25%(group Ⅰ), 14.33%(group Ⅱ), 15.53%(group Ⅲ) and 16.60%(group Ⅳ), respectively.The experiment lasted for 60 days, 30 days for the early stage and 30 days for the later stage;And on the 30th and 60th day, blood samples were collected from one head sheep chosen from each replication, and at the end of the feeding test, 3 lambs were randomly selected from each experiment group for a 15 days digestion and metabolism experiment.The results showed that ADFI and ADG in the early and total trial period were significant differences(P<0.05), ADFI and ADG of group Ⅲ were higher than others.The apparent digestibility of nutrients and ME/DE were not significantly changed(P>0.05).In the detection of serum biochemical indexes, there was no significant difference in the earlier stage(P>0.05);At the end of the test, PK and CK showed significant difference between groups(P<0.05).PK presented a rising trend as the protein level improved, CK of group Ⅰ was higher than other groups.In Super Merino lamb early weaning diet, 15.53% protein level in the diet could significantly improve ADG, the apparent digestibility of nutrients, metabolic energy digestibility.At the end of the test, PK and CK were affected by dietary crude protein level significantly.     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

多型FMDV VP1表位嵌入型载体pMG-SLPMultiVP1的构建及其表达产物鉴定

构建出表达载体pMG-SLPMultiVP1,并对其进行双酶切鉴定及PCR鉴定。将阳性重组质粒转入到发酵乳杆菌中进行表达,并对表达产物进行SDS-PAGE、Western blot和IFAT鉴定。多型FMDV VP1表位嵌入的SLPMultiVP1融合基因成功克隆入穿梭质粒pMG36e中,其表达产物大小约为35ku,部分表达产物位于发酵乳杆菌表面。成功构建了一个乳杆菌嵌入型表达载体pMG-SLPMultiVP1,为以发酵乳杆菌为活菌载体的表位疫苗研究奠定了基础。