禽流感血清抗体与卵黄抗体消长规律比较研究

应用禽流感病毒二价（H5、H9）油乳剂灭活疫苗免疫160日龄非免疫蛋鸡，免疫接种后分别于第0、7、14、21、28、35 d采集相应的鸡蛋和血清，采用血凝抑制(HI)试验监测血清及卵黄中H5、H9抗体的消长规律，结果表明：H5、H9血清抗体于免疫后7 d时开始产生，14 d时到中等水平，21 d时达到最高值，一直维持到35 d之后；而H5、H9卵黄抗体与血清抗体水平相比相对滞后7 d左右；卵黄抗体与血清抗体在28 d时均达到高峰值并一直维持到35 d之后，28 d后血清和卵黄抗体达到相同水平。本研究为临床上以禽流感卵黄抗体检测替代血清抗体检测及以后禽流感高免卵黄抗体的研究提供了依据和数据。     

DNA疫苗的作用机制及新型免疫策略的研究进展

疫苗是预防和控制严重传染病的重要手段，将基因工程用于疫苗生产已经成为生物技术的热点内容之一。治疗性基因工程疫苗是近些年研究的重点，并迅速从治疗传染性疾病的研究扩展到非传染性疾病的研究，如治疗性乙肝疫苗、治疗性肿瘤疫苗等。本文针对这一热点阐述基因工程DNA疫苗的作用机制及新型免疫策略研究的进展。     

斯氏艾美耳球虫的致病性及病理学研究

用纯种斯氏艾美耳球虫孢子化卵囊人工感染25只断奶幼兔,进行致病性和病理学研究。试验兔分为1个不感染对照组和4个感染组,各感染组每只幼兔分别口服感染2.0×103、1.0×104、5.0×104、2.5×105个孢子化卵囊。与对照组相比,接种2.0×103个和1.0×104个孢子化卵囊的两个感染组的平均病变记分、肝重指数、血清转氨酶活性有显著差异(P<0.05);接种5.0×104个和2.5×105个孢子化卵囊的两个感染组上述指标有极显著差异(P<0.01)。表明斯氏艾美耳球虫对幼兔具有很强的致病性,能导致严重的肝球虫病。     

绵羊体外成熟卵母细胞OPS法玻璃化冷冻保存试验

研究以EDFS30为玻璃化冷冻液,以卵母细胞解冻后孤雌激活和体外受精后的卵裂率、囊胚发育率作为评价指标,探讨了以OPS法玻璃化冷冻保存体外成熟绵羊卵母细胞的效果。结果表明:卵母细胞孤雌激活后的卵裂率,冷冻组(64.2%)显著(P<0.05)低于毒性组(76.7%)和对照组(79.1%),而毒性组和对照组无显著(P>0.05)差异;卵母细胞孤雌激活后的囊胚发育率,冷冻组(4.2%)和毒性组(5.8%)均显著(P<0.05)低于对照组(20.2%),毒性组和冷冻组无显著(P>0.05)差异;冷冻组和毒性试验组卵母细胞体外受精后的卵裂率和囊胚发育率(67.6%和7.1%;62.3%和9.1%)均显著低于对照组(78.4%和28.4%)(P<0.05),而毒性组和冷冻组无显著(P>0.05)差异。可见以EDFS30为玻璃化冷冻液,采用OPS法冷冻保存绵羊体外成熟卵母细胞会在一定程度上降低其受精能力和胚胎发育能力。     

新城疫病毒F、NP、M和HN基因在昆虫细胞内的共表达

为构建杆状病毒转移载体，通过PCR的方法将F48E9株新城疫病毒F基因上的StuⅠ位点和NP基因上的XbaⅠ位点进行突变，扩增出全长的F、NP以及F48E9株新城疫病毒M和HN基因片段，将其克隆到pMD18-T载体，再将F、NP、M和HN基因依次亚克隆到杆状病毒转移载体pAeAB4上，F基因和M基因均在p10启动子的操控之下，NP基因和HN基因构建到两个polyhedrin启动子下游，构建重组杆状病毒转移载体pAeAB4-F-NP-M-HN。pAeAB4-F-NP-M-HN与线性化的杆状病毒DNA共转染昆虫细胞Sf9，获得重组杆状病毒rBae-F-NP-M-HN。重组杆状病毒感染Sf9细胞，72h后收集细胞和培养上清。Western blot分析显示：M、NP、F和HN蛋白在培养上清中得到了共表达，大小与预期结果一致；感染细胞内只检测到了HN蛋白的表达。这表明M、NP、F和HN蛋白在昆虫细胞内共表达可以自我装配成病毒样颗粒，并且以出芽的方式释放到培养基中。该重组杆状病毒的获得为研究新城疫病毒各结构蛋白之间的相互作用和确定病毒粒子出芽的驱动力等方面奠定了基础。     

紫色辣椒新品种‘紫燕1号’

 ‘紫燕 1 号’为辣味中等彩色辣椒一代杂种。利用国内优良自交系与从国外紫色甜椒多代自交优良株系杂交而成。该品种早熟, 从定植至采收商品椒约42 d; 抗性强, 耐低温弱光; 果实牛角形, 鲜果紫色, 生物学成熟果深红色, 鲜椒产量39 000～45 000kg/hm²。适于春早熟和秋延后保护地栽培。     

Protective effect of atrial natriuretic peptide on alveolar type II cells

AIM: To study the protective effect of atrial natriuretic peptide (ANP) on alveolar type II cells (AT-Ⅱ) damaged by lipopolysaccharide (LPS).METHODS: AT-Ⅱ were placed in a 6 well cell culture cluster (0.5×106 cells/cm2) and divided into 3 groups: (1) control group (n=6), the medium consisted of RPMI-1640 without FBS. (2) LPS group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L). (3) ANP group (n=6), the medium consisted of RPMI-1640 without FBS supplemented with LPS (1 mg/L) and ANP (10-8, 10-7, 10-6 mol/L). After 4, 12 and 24 h, the cell culture mediums of control group, LPS group and ANP (10-7 mol/L) group were collected, and those of the ANP (10-6, 10-8 mol/L) group were collected after 12 h. Alkaline phosphatase(AKP), lactate dehydrogenase(LDH), malondialdehyde(MDA), total phospholipids (TPL) and surface tension (ST) in the medium of every group were examined. RESULTS: AT-Ⅱ were characterized by AKP staining. The contents of LDH, AKP and MDA in the medium of every ANP group were lower than those in the corresponding LPS group. The TPL content in the medium of every ANP group was higher than that in the corresponding LPS group, and the change of ST of the medium was opposite to that of TPL. The effect at 12 h was the most significant, for example, at 12 h, the activities of AKP in the mediums were: control (43.5±10.4) U/L, LPS (98.1±16.4) U/L, LPS+ANP (10-6) (46.4±10.5) U/L, LPS+ANP(10-7) (60.7±9.5) U/L, LPS+ANP(10-8) (91.3±13.9) U/L.CONCLUSION: ANP protects the AT-Ⅱ from being damaged by LPS and promotes the secretion of pulmonary surfactants.     

Screening differentially expressed genes involved in apoptosis of primary cultured rat cerebellar granule neurons by mRNA differential display RT-PCR

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured.Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs.ESTs were subcloned into pGEM-T EasyTM vector and then sequenced.Alignment assay in non-redunant database was applied for encoding information.Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR.17 of them were subcloned and sequenced.5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting.CONCLUSION: DD-PCR is a rapid,simple-operation and sensitive method for screening differentially expressed genes,which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.     

Sequence analysis and prokaryotic expression system construction of PIA genes isolated from Neisseria gonorrhoeae

AIM: To analyze the nucleotide and putative amino acid sequences of PIA genes isolated from N.gonorrhoeae and to construct the prokaryotic expression system of PIA gene.METHODS: The entire PIA genes from 9 strains of N.gonorrhoeae were amplified by using high fidelity PCR.The target amplification fragments were sequenced after T-A cloning.Homology comparison of the nucleotide and putative amino acid sequences of PIA genes from the isolates with the reported sequences in GenBank was then performed.A prokaryotic expression system of PIA gene was constructed.Different dosages of IPTG were applied to induce the expression of the target recombinant protein (rPIA) and 10% SDS-PAGE plus Bio-Rad Agarose Image Analysor was used to determine the expression level of rPIA.rPIA was extracted using Ni-NTA affinity chromatography and the purified effect was detected by SDS-PAGE.RESULTS: In comparison with the reported PIA gene sequences (GenBank No: L19962),the homologies of nucleotide and putative amino acid sequences of PIA genes from the isolates were 99.6%-100% and 99.1%-100%,respectively,which indicated that all the isolates were belonging to serovars IA6.Output of rPIA was as high as 50.1% of the total bacterial proteins.The purified rPIA only showed a single target protein fragment in gel.CONCLUSION: Serovar IA6 is dominant in the local N.gonorrhoeae isolates and sequences of the encoding gene are relatively conserved.The constructed prokaryotic expression system is able to express rPIA with high efficiency,which may lay a foundation for further development of serological detection kit and vaccine of N.gonorrhoeae.