Sequence Analysis and Prokaryotic Expression of NSP1 Gene of Bovine Rotavirus UK Strain

Rotavirus is a major cause of acute diarrhea in both many kinds of young animals and children under 5 years old.Rotavirus NSP1, a 55 ku RNA binding protein, is the product of gene 5, which can subvert innate immune responses and be one of virulent determinant factors.According to the sequence in GenBank, specific primers targeting to NSP1 gene were designed and the gene was amplified by RT-PCR, following by being cloned into the pET-28a(+) vector.It showed that the full length of NSP1 gene was 1 473 bp, encoding 491 amino acids.The NSP1 shared the highest identity with WC3 strain.The recombinant protein was induced in E.coli Rosetta(DE3) by IPTG and was analyzed by SDS-PAGE and Western blotting.The results revealed that NSP1 recombinant protein existed in the form of inclusion body with the molecular weight of 55 ku.The purified recombinant protein could be recognized by His-tag antibody.This study laid the foundation for further research on the relationship between the intracytoplasmic location of NSP1 protein and its activity.     

公猪精液PRRSV变异株检测及NSP2、ORF5基因序列分析

用荧光RT-PCR方法从某猪场精液中检测到猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)变异株(NSP2 1 594～1 680 bp缺失)核酸阳性.提取1份阳性样品病毒核酸测定和分析其NSP2和OBF5全基因序列,结果表明NSP2基因由950个氨基酸组成,与CH-1a、VR-2332等经典PRRSV相比481位缺失1个氨基酸、532～560位连续缺失29个氨基酸,与JXA1、HUN4等毒株具有相同的缺失特性;ORF5基因第13、151位为具有强毒特性的精氨酸(R),存在4个潜在的糖基化位点,分别位于30～32、35～37、44～46和51～53位氨基酸.遗传进化分析表明,测定序列与JX-A1、HUN4等毒株关系最近,与CH-1a、NVSL等同属一个大分支,而与BJ-4、P129、RespPRRS MLV、VR-2332等处于不同分支.研究证实公猪精液可携带PRRSV变异株,因此猪场(群)在人工授精和引进精液时必须强化检疫和生物安全措施.     

The fermentative capacity of growing pigs and adult sows fed diets with contrasting type and level of dietary fibre

Adult sows have a more developed gastro-intestinal system than young growing pigs resulting in a superior capacity to digest fibrous components. The main objective of this study was to describe the capacity of sows and growing pigs to digest the fibre components in concentrated low dietary fibre (DF) diet and two high DF diets similar in DF but with contrasting DF solubility. Six sows and four growing pigs were fitted with a simple T-cannula at the terminal ileum. Each balance experiment consisted of a 7-day initial period followed by a 3-day collection of faeces and a 2 or 3-day collection of ileal digesta for growing pigs and adult sows, respectively. When feeding diets with high proportions of soluble non-starch polysaccharides (NSP), the difference in fermentation capacity between growing pigs and adult sows was small. The adult sows showed a higher fermentative capacity, evidenced by a higher fermentation and a higher methane production, when high fibre diet with high amount of insoluble fibre was fed.     

香蕉束顶病毒海口分离物NSP基因的原核表达

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆NSP (Nuclear shuttle protein)基因的编码区,并通过Gateway技术定向重组到原核表达载体pDEST~(TM)-17中的6xHis标签下游,经菌落PCR和测序鉴定结果表明,成功构建了原核表达载体pDEST~(TM)-17-NSP.阳性克隆转化Ecoli BL21(DE3),经IFFG诱导表达、SDS-PAGE分析和western blot检测,结果显示,融合蛋白以包涵体的形式稳定表达,分子量约为20 ku,与预计值相符.通过诱导条件的优化,确定了最佳的融合蛋白表达条件为25℃、0.1 mmol/L IPTG条件下诱导4h.从而为制备NSP多克隆抗体和进一步研究NSP的功能奠定了基础.     

猪传染性胃肠炎病毒NSP8蛋白单克隆抗体的制备及抗原表位的初步鉴定

为鉴定猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)NSP8蛋白的抗原表位,本研究对GST-NSP8重组蛋白进行了原核表达,并用纯化后的重组蛋白免疫6周龄BALB/c小鼠,取免疫小鼠脾脏,采用常规杂交瘤细胞融合方法,经3次亚克隆后制备了1株稳定分泌抗NSP8蛋白的单克隆抗体杂交瘤细胞株。分泌的单克隆抗体亚类鉴定其重链为IgG1型,轻链为κ链;杂交瘤细胞培养上清的效价为1∶3 200。Western blotting试验结果表明该单克隆抗体能识别原核及真核表达的NSP8重组蛋白。利用截短表达的方法对NSP8蛋白进行抗原表位的鉴定,初步确定了单克隆抗体针对的抗原表位序列为31SPQILKQLTKAFNIAKSDFEREASV55。本研究制备的单克隆抗体及对抗原表位的鉴定,为TGEV NSP8蛋白相关功能的研究奠定了基础。     

小麦日粮添加非淀粉多糖酶制剂对雏鸡生长及血液中血糖、尿酸和某些激素水平的影响

2个系列试验研究小麦日粮添加非淀粉多糖酶制剂对雏鸡生长及血液中血糖、尿酸和某些激素水平的影响。试验1将7日龄雏鸡72羽随机分为3组,即小麦基础日粮组、小麦日粮加0.15％浙江酶-1组和小麦日粮加0.15％赤峰酶组。试验2将7日龄雏鸡248羽,随机分为8组,即玉米基础日粮组、小麦基础日粮组、小麦日粮加0.1％、0.2％、0.5％浙江酶-2组和小麦日粮加0.1％、0.2％、0.5％芬兰酶组。试验1结果表明,和小麦组相比,添加0.15％浙江酶-1组雏鸡增重显著提高(P<0.05),料重比下降但不显著(P>0.05);而添加0.15％赤峰酶组增重提高,料重比下降,但均未达到显著(P>0.05)。试验2结果表明,和小麦日粮组相比,添加浙江酶-2各组雏鸡增重提高9.3％～15.7％(P<0.01),料重比降低6.3％～10.8％(P>0.05);添加芬兰酶各组雏鸡增重提高6.2％～9.7％(P<0.05),料重比降低4.2％～13.3％(P<0.05);添加酶各组和玉米组相比,雏鸡增重无明显差异。试验1和试验2血液指标测定结果表明,小麦日粮添加酶制剂可使雏鸡T4、IGF-1水平显著提高(P<0.05),对T3、胰岛素、血糖和尿酸含量未发现明显影响(P>0.05),提示酶制剂可以通过影响机体的代谢激素促进雏鸡生长。     

Combined effect of using near‐infrared spectroscopy for nutritional evaluation of feed ingredients and non‐starch polysaccharide carbohydrase complex on performance of broiler chickens

This study was carried out to evaluate the combined effect of using near‐infrared spectroscopy (NIRS ) for nutritional evaluation of feed ingredients and the addition of non‐starch polysaccharide carbohydrase complex (NSP enzymes) on the growth performance of broilers fed diets produced with low‐quality wheat and soybean meal. A 2 × 2 trial design was performed, with seven replicates of 40 male Ross 308 broilers per treatment, evaluating the effect of the addition of NSP enzymes and the ingredients’ nutritional matrix based on table values or NIRS values. Diets without added enzymes were formulated to reach nutritional requirements, whereas diets with enzymes were reformulated, reducing the apparent metabolizable energy (AME ) by 85 kcal/kg. In the overall period (days 0–35), broilers fed diets formulated using NIRS values had higher (P  < 0.001) average daily gain (+11.3%) and daily feed intake (+7.2%), and a lower (P  < 0.001) feed conversion ratio (?5.3%) compared to those fed diets formulated using table values. When formulating diets for broilers with low‐quality feed ingredients, performance can be improved by considering NIRS values and by the addition of NSP enzymes, even with a reduction of AME . These nutritional approaches are efficient in improving broilers’ performances by themselves and even more so when they are combined.     

猪繁殖与呼吸综合征病毒NSP2抗原表位区的原核表达及免疫原性分析

试验旨在表达纯化猪繁殖与呼吸综合征病毒NSP2重组蛋白。利用PCR技术扩增获得大小为750 bp的NSP2 226－475位氨基酸编码序列,然后将该序列亚克隆到pET-28a（+）表达载体上,通过pET-28a（+）表达载体在大肠杆菌Rosetta-gami 2（DE3）pLys中得以表达,获得大小为29 ku的重组蛋白,对重组蛋白诱导时间进行优化,SDS-PAGE显示重组蛋白在37 ℃经1 mmol/L IPTG诱导8 h时表达量最大,免疫印迹结果证实表达的蛋白能被PRRSV阳性血清识别,具有良好的免疫原性。本研究成功表达纯化了NSP2重组蛋白,为目标蛋白作为包被抗原建立检测PRRSV血清抗体的ELISA方法奠定了基础。     

NSP酶制剂对生长鹅小肠食糜黏度及盲肠微生物数量的影响

选择300只29日龄生长鹅进行28 d的饲养试验,研究在高纤维基础日粮中分别添加0.2%和0.4%的NSP酶制剂对生长鹩小肠食糜黏度及盲肠微生物数量的影响.对小肠食糜黏度的研究结果显示:0.2%与0.4%的NSP酶制剂对十二指肠食糜相对黏度的下降幅度分别为9.43%和10.69%(P<0.05),对空肠食糜相对黏度的下降幅度分别为10.74%和12.81%(P<0.05),对回肠食糜相对黏度的下降幅度分别为14.11%和15.03%(P<0.05).对盲肠微生物数量的研究结果显示:0.2%与0.4%的NSP酶制剂对盲肠大肠杆菌、双歧杆菌和乳酸杆菌均无显著影响(P>0.05).     

新疆猪繁殖与呼吸综合征病毒分离鉴定及Nsp2和ORF5基因遗传变异分析

为探索新疆猪繁殖与呼吸综合征病毒（PRRSV）的变异特征,利用RT-PCR、ORF5 和部分NSP2 基因序列进行分析,鉴定从新疆某猪场疑似猪高热病死亡猪肺脏分离出的一株病毒。分离的病毒株被鉴定为PRRSV 美洲型,命名为XJ-Q 株。该株病毒与香港分离株HK13 亲缘关系最近,与国内变异株（HUB1 和JXA1）同源性较为亲近。与美洲型标准毒株VR-2332 相比,该分离株在481 位和532~560 位共有30 个氨基酸缺失;与国内分离的PRRSV 变异株相比,在487~489 位有3 个氨基酸的独特缺失。结果表明,新疆分离株为PRRSV 美洲型,属于新缺失的PRRSV毒株。