气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。     

拷贝数变异在畜禽中的研究进展

拷贝数变异（copy number variation,CNV）具有多种形式的变异结构,在品种多样性、生物进化和疾病相关性等研究中起着重要作用,并具有片段长度大、覆盖范围广等特点。随着分子生物学的发展及DNA测序技术的日渐成熟,人们对遗传变异的研究不断向DNA分子水平深入,多态性标记在畜禽育种中已逐渐成为动物育种研究的趋势和主流。由于CNV对基因的调控和表达所造成的影响更为显著,因此,CNV在重要畜禽中的研究越来越多。目前,已检测出大量有关畜禽重要经济性状的基因序列变异,并有许多研究均表明CNV与动物的重要经济特征及疾病的发生有关。笔者主要通过参考国内外相关的研究报道,简述了CNV的相关研究背景、概念、突变机制,归纳总结了CNV对牛、羊、猪、鸡的经济性状、繁殖性状和疾病调控的影响,以期通过对这些重要畜禽的基因组学研究揭示其适应性遗传机理和表型性状差异的遗传基础,开发相应的分子遗传标记,为畜禽的标记辅助选育提供理论基础。     

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.     

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.     

Screening of Probiotics Inhibiting E. coli K88 in vitro

The probiotics tested in the experiment were isolated from the intestinal of weaning piglets.The isolated probiotics and E.coli K88 were inoculated into the culture of intestinal porcine epithelial cell-1 (IPEC-1).The activity of lactate dehydrogenase (LDH) in the supernatant was measured after incubating for 2.5 h.At the same time, the probiotics and E.coli K88 were co-cultured in vitro, the number of E.coli K88 was counted and the probiotics which could be resistant to the E.coli K88 were selected 2.5 h later.The results showed that the Lactobacillus casei and Bacillus coagulans could significantly reduce LDH activity (P<0.05), decrease the damage of E.coli K88; Bacillus coagulans could inhibit the growth of E.coli K88.At the same time, Bacillus coagulans could resist high temperature, acid and bile salt.The results showed that Bacillus coagulans strains had great potential as the application of probiotics strains.The methods could be used as a model of screen probiotics which could inhibit the growth of E.coli K88 in vitro.     

Cloning and Sequence Analysis of HA and NA Genes of One Strain of H6N6 Subtype Avian Influenza Virus Isolated from Guizhou Province

To investigate the epidemic situation of H6N6 subtype avian influenza virus (AIV) in Guizhou province,A/duck/Guizhou/013/2014 was isolated from Sansui duck in live poultry market of Guizhou in 2014,the hemagglutinin (HA) and neuraminidase (NA) genes of DK/GZ/14 were subjected to clone and sequence analysis.The results showed that HA gene had the highest nucleotide homologies (97.5%) with the duck-origin H6N6 subtype AIV isolated from Eastern China in 2009,and the strains of HA gene proteolytic cleavage sites was P-Q-I-E-T-R-G,which accordeol with the molecular characteristic of low pathogenic AIV (LPAIV).However,NA gene of A/duck/Guizhou/013/2014 had the highest nucleotide homologies (98.2%) with the duck-origin H6N6 subtype AIV isolated from Fujian in 2007.The phylogenetic tree showed that A/duck/Guizhou/013/2014 and Hunan strains located in the same branch,while three duck-origin H6N6 subtype AIV isolated from Guizhou in 2007 and A/duck/Guizhou/013/2014 located in the different branch for HA and NA genes in genetic evolution,which suggested that A/duck/Guizhou/013/2014 was far with the local H6N6 subtype.The results also clearly indicated that duck-origin H6N6 subtype AIV had genetic diversity in duck population in Guizhou.     

高温胁迫对丹参光合生理指标及叶绿素荧光特性的影响

以丹参为试材,采用土培盆栽法,于第1茬花开前进行高温胁迫处理,研究了高温胁迫对丹参生理指标、光合及叶绿素荧光特性的影响。结果表明:随着高温胁迫时间的延长,丹参叶片超氧化物歧化酶(SOD)活性呈上升趋势,而过氧化氢酶(CAT)活性呈下降趋势;丙二醛(MDA)、游离脯氨酸(Pro)含量均呈上升趋势,且与胁迫时间呈极显著多项式关系。净光合速率(Pn)、气孔导度(Gs)、胞间二氧化碳浓度(Ci)和蒸腾速率(Tr)均呈下降趋势,且各指标均与胁迫时间呈负相关。最大光化学量子产量(Fv/Fm)、PSⅡ潜在光化学活性(Fv/Fo)、有效光化学量子产量(Fv′/Fm′)、光化学淬灭系数(qP)和表观光合电子传递速率(ETR)均呈下降趋势,且各指标均与胁迫时间呈负相关,非光化学淬灭系数(NPQ)呈先上升后下降趋势。研究表明,高温(40℃)对丹参生理指标、光合及叶绿素荧光特性有较大的影响,且各功能指标共同作用,以减轻植株所受伤害。     

不同系统梨品种抗旱性初步评价

以5大梨系统20个梨品种的2年生盆栽梨苗为试材,研究水分胁迫及复水条件下膜脂过氧化物质、渗透调节物质及抗氧化酶活性的变化特征。结果表明,秋子梨系统梨品种具有较高的净光合速率、气孔导度和水分利用效率,较低的蒸腾速率和叶面饱和蒸汽压亏缺,抗旱性强于其他品种;砂梨抗旱性最弱。随着水分胁迫时间的延长,抗旱性较弱的西洋梨和砂梨MDA和O_2~-含量迅速增加,膜系统受到严重伤害。西洋梨和白梨随着干旱胁迫的加重脯氨酸大量积累,启动渗透调节来缓解水分胁迫造成的伤害。水分胁迫前期不同系统梨品种SOD和POD活性呈持续上升趋势,胁迫过重则活性降低,且抗氧化酶增加的幅度与梨品种的抗旱性呈正相关关系。综合评价认为,秋子梨、新疆梨及白梨系统品种的抗旱性较强,适宜在干旱、半干旱地区发展,西洋梨和砂梨则抗旱性较弱,适宜在湿润、半湿润地区发展。     

一类二维加权积分数值求积方法及其误差分析

针对一类二维加权积分的数值计算方法及误差分析进行研究,参照一维广义高斯积分数值求积方法提出一种具有较高代数精度的计算方法并对其误差做分析,同时给出几个简明的结果。这些结果可用到诸如小波分解,图像处理与多分辨数字信号处理等领域。     

浮床种植草坪草对富营养化水体的净化作用研究

采用浮床种植草坪草技术,研究了高羊茅Festuca arundinacea、匍匐翦股颖Agrostis stolonifera 2种草坪草对富营养化水体的净化能力.试验结果表明,2种草坪草对富营养化水体中的氮(N)、磷(P)、化学需氧量(CODcr)等具有明显的净化效果,尤其对总磷(TP)的净化能力最强,能有效改善富营养化水体的水质;2种草坪草之间,匍匐翦股颖对TP的去除效果显著高于高羊茅对TP的去除效果.研究结果显示浮床种植草坪草技术可能是一条有效的修复富营养化水域的新途径.