新城疫病毒结构蛋白功能及检测技术研究进展

新城疫是一种由新城疫病毒引起的烈性传染病,病死率很高,严重危害家禽养殖业,被世界动物卫生组织列为必须报告的动物传染病。在我国,新城疫的暴发和流行虽然已得到了控制,但病毒的变异给新城疫的防控工作带来了困难。新城疫病毒的隐性感染也对我国禽类产品的质量及出口造成了一定影响。因此,明确新城疫病毒各结构蛋白的功能、改进现有检测技术以及开发新的检测技术平台是目前新城疫防控工作的重点。论文对新城疫病毒结构蛋白功能及检测技术的进展进行了系统的阐述。     

猪繁殖与呼吸综合征病毒GP5、M蛋白基因的克隆及其基因疫苗构建

参照GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲型代表株VR2332 GP5和M蛋白基因序列,设计并合成2对引物,用RT-PCR方法分别扩增出PRRSV野毒株GP5和M蛋白基因603,525bp片段,并将其分别克隆到pMD18-T载体。测序正确后,将GP5和M蛋白基因分别克隆到真核表达载体pEGFP-C1上,成功构建基因疫苗表达载体pEGP5-C1和pEM-C1。小鼠免疫试验证实,这些基因疫苗质粒可以诱导小鼠产生特异性抗体,并在二免后1周开始检测到特异性淋巴细胞增殖反应。     

Study on Proliferation and Apoptosis of EBL Cells Induced by P48 Protein of Mycoplasma bovis

The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung (EBL) cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10 μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation (P<0.01),while 0.1 and 0.5 μg/mL protein concentration had no inhibitory effect (P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.     

Ghrelin对雌激素诱导小鼠胸腺萎缩过程中细胞因子及凋亡相关蛋白基因表达的影响

应用光镜和实时定量PCR技术研究了Ghrelin对雌激素诱导小鼠胸腺萎缩过程中胸腺形态学以及部分细胞因子和凋亡相关蛋白基因表达的影响。结果显示,注射Ghrelin后,雌激素诱导的小鼠萎缩胸腺在形态学上基本恢复到正常水平,胸腺中IL-6、TGF-[31、Caspase3、FADDmRNA含量显著降低,Caspase9、FasLmRNA含量略为下降,而IL-7、Bcl-2mRNA含量有所上升。结果表明,Ghrelin可能通过促进胸腺上皮细胞的增殖以及阻断Caspase级联程序和Fas／FasL凋亡信号通路抑制胸腺细胞的凋亡,从而逆转雌激素诱导的小鼠胸腺萎缩。     

昆虫共生菌沃尔巴克氏体(Wolbachia)的wsp基因密码子使用模式分析

沃尔巴克氏体(Wolbachia)主要感染昆虫等节肢动物,是一类革兰阴性共生细菌。为探讨昆虫纲共生菌Wolbachia的系统发育,研究Wolbachia基因组中进化速度较快的外膜蛋白基因wsp的密码子使用模式及影响因素,统计分析该基因的GC含量(GCall、GC1、GC2、GC3、GC3s)、有效密码子数(ENc)、相对同义密码子使用度(RSCU)等指标。结果显示:昆虫纲共生菌Wolbachia的wsp基因密码子偏好性均不强,昆虫纲不同目间共生菌Wolbachia的wsp基因碱基组成及ENc值差异较小;多数基因数据点沿标准曲线或在其附近分布,突变对碱基组成的影响较弱;18种由多个密码子编码的氨基酸中有11种氨基酸的偏好性密码子在昆虫纲7个目及对照蛛形纲1个目间均相同,有2种氨基酸的偏好性密码子在7个目间相同,这些密码子均以A/U结尾;供分析的140个wsp基因中仅编码6个半胱氨酸(Cys);对应分析中第1、第2向量轴贡献率均不高,分别为13.53%和12.49%,均与碱基组成(GC1、GC2、GC3)显著相关。综合各项分析认为,Wolbachia的wsp基因密码子偏好性不具有宿主分类特异性,密码子使用模式主要受碱基组成影响,而碱基组成主要受选择影响。     

Serum protein electrophoresis in retired racing Greyhounds

BACKGROUND: Retired racing Greyhounds are becoming common as pets. Because of their unique physiology, results of routine laboratory tests are frequently outside the reference interval for dogs. Compared with other breeds, Greyhounds have low serum protein concentrations, but the concentrations of different serum protein fractions have not been reported. OBJECTIVES: Our objectives were to evaluate the results of serum protein electrophoresis (SPE) in healthy, retired racing Greyhounds and compare them with a control group of age- and gender-matched non-Greyhound dogs. METHODS: Agarose gel electrophoresis was done using a standard method; the gels were stained with amido black and scanned with a Cliniscan 2 densitometer (Helena Laboratories, Beaumont, TX, USA). Protein fractions were identified by visual inspection of the electrophoretogram. A Student's t-test assuming equal variances was used to compare the concentration of the different fractions between groups. RESULTS: The concentrations of total protein, total globulins, and alpha-1-, alpha-2-, beta-1-, and beta-2-globulins were significantly lower and the albumin to globulin (A:G) ratio was significantly higher in Greyhounds than in non-Greyhound dogs (P < .05). There was no significant difference in albumin or gamma-globulin concentrations. CONCLUSIONS: Low serum protein concentrations in Greyhounds are the result of low concentrations of a- and b-globulins. These results should be kept in mind when evaluating both healthy and sick Greyhounds. Additional studies are needed to identify the individual proteins associated with low alpha- and beta-globulin concentrations in Greyhounds.     

不同粗蛋白水平日粮对种公鸡繁殖性能的影响

试验以21周龄新扬州种公鸡为对象,研究了不同粗蛋白水平日粮对种公鸡繁殖性能的影响。结果表明,粗蛋白质水平为14%的B组公鸡精液量、精子活力和精子密度均明显地高于粗蛋白水平为11%的A组和粗蛋白质水平为17%的C组,而精子畸形率为B组最低(P>0.05);日粮粗蛋白水平对成年种公鸡的睾丸性状影响不大(P>0.05),而年龄对公鸡的睾丸性状有着显著的差异(P<0.05);A组的血浆中睾酮T、促黄体素LH和促卵泡素FSH含量分别比B组高了6.34%、19.64%和3.88%,比C组高了7.20%、17.54%和9.84%,差异不显著(P>0.05)。     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus

Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A.     

The effects of continuous and rotational grazing of sourveld on the quality of herbage selected by Simmentaler heifers

A grazing trial was conducted at the Kokstad Agricultural Station to compare the dry matter digestibility (DMD) and crude protein content (CP) of diets selected by four fistulated cattle which were continuously and rotationally grazing sourveld. Two stocking rates of 1,0 and 2,3 animals/ha (0,5 and 1,2 LSU/ha) were applied under each grazing method. The mean DMD and CP values of diets selected in the continuous and rotational grazing treatments, respectively, were: DMD=64,3%, 62,3%; CP=6,63%, 6,88%. The effect of stocking rate on the quality of the diet selected varied between the two grazing methods. DMD and CP of diets selected under rotational grazing decreased with time during the week‐long period of stay in each paddock. On average, DMD and CP values on the first and sixth day respectively, were: DMD=66,6%, 60,5%; CP=8,37%, 6,01%. These differences were more pronounced at the higher than at the lower stocking rate. It was concluded that, although some of the treatment differences were small, grazing method differences in the DMD of diets might at least partially account for observed differences in livemass gains under the two grazing systems.