烟草蚀纹病毒外壳蛋白基因的克隆及在毕赤酵母中的表达

摘 要:利用RT-PCR方法获得烟草蚀纹病毒（TEV）外壳蛋白（CP）基因,大小为789 bp。经过EcoRI和NotI双酶切、定向克隆到pPIC9K,构建了真核表达载体pPIC9K-TEVCP。将重组质粒用Sal I线性化后电转化导入毕赤酵母GS115中, 经PCR鉴定为阳性的菌落,利用甲醇进行诱导表达,筛选出了高效表达菌株GS115-11和GS115-14,表达的蛋白大小约为37 kD。表达产物经SDS-PAGE割胶纯化后,免疫家兔,制备了特异性抗血清,ELISA法检测效价为1:1500。Western blot结果显示,制备的抗血清可以用来检测田间的发病植株。      

人甲胎蛋白AFP在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21(DE3)中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

人甲胎蛋白 AFP 在大肠杆菌中表达和鉴定

本研究通过应用基因工程技术,重组构建编码人全长甲胎蛋白AFP基因的原核表达载体pET22b-AFP,并将其转化到大肠杆菌宿主菌BL21（DE3）中进行诱导表达目的蛋白AFP。采用PCR法扩增编码AFP蛋白的cDNA序列片段,并将其克隆到含有His-tag的pET22b原核表达载体上;重组质粒经双酶切及测序鉴定正确后转化到大肠杆菌BL21（DE3）中进行IPTG诱导表达;对诱导过程中不同浓度的IPTG和不同的温度进行优化;选定最佳的优化条件进行诱导表达目的蛋白;SDS-PAGE电泳和Western Blot法鉴定表达产物。结果表明,通过PCR扩增技术获得了编码AFP蛋白的基因片段;重组质粒经双酶切和基因测序等方法鉴定后,确认了重组质粒已经构建成功;表达产物通过利用SDS-PAGE和Western Blot法检测到在66 kDa附近出现条带,与预期值相符,表明重组AFP蛋白已成功表达。     

胶体金免疫层析法快速检测李痘病毒的研究

用RT-PCR的方法克隆李痘病毒的外壳蛋白(CP)基因,然后将其连接到原核表达载体p ET29(a)上,得到p ET29a-PPV重组载体,然后将该载体转化大肠杆菌BL21(DE3),经过IPTG诱导PPV CP基因在大肠杆菌中得到了高效表达。回收表达蛋白后免疫家兔,获得PPV特异性抗血清,经过酶联免疫吸附法测定其效价达到3.2×104,然后采用该抗血清制备出专门检测李痘病毒的胶体金免疫试纸条,测试结果表明,该试纸条不仅特异性强、稳定性好,灵敏度也高,可以检测到1μg/m L粗提病毒和稀释100倍的病汁液,操作简便,10分钟之内即可出检测结果,特别适宜口岸检疫和田间快速诊断。     

蜂毒肽与死亡素杂合肽(MT)在大肠杆菌中融合表达的研究

人工合成的蜂毒肽与死亡素的杂舍肽DNA序列，经聚合酶链式反应（PCR）扩增后得到带有BamHI和XhoI限制性酶切位点的序列．将此基因克隆至载体pET-32a，成功构建了重组质粒pET32a—MT．测序验证后转化大肠杆菌BL21（DE3），37℃IPTG诱导，获得高效表达，融合蛋白经Ni柱亲和纯化后用肠激酶切下杂合肽，杯碟法检验酶切产物具有抑菌活性。     

阪崎肠杆菌α-葡萄糖苷酶基因克隆、表达及活性研究

目的:利用pET质粒原核表达体系克隆、表达阪崎肠杆菌α-葡萄糖苷酶基因,表达产物进行Ni-NTA柱纯化,利用α-葡萄糖苷酶分解4-硝基苯基-α-D-呋喃型葡萄糖的特性进行表达纯化合的蛋白活性鉴定,为进一步制备阪崎肠杆菌检测用单克隆抗体奠定了基础.方法:从阪崎肠杆菌ATCC29544标准菌株中克隆获得阪崎肠杆菌α-葡萄糖苷酶基因,连接pET22b( )表达载体后转化大肠杆菌BL21(DE3)菌株,利用不同浓度的异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达外源基因,分别检测不同诱导时间、不同浓度IPTG作用下表达产物,筛选高表达菌株.表达菌株大量培养后,超声波及蛋白酶K作用破菌,Ni-NTA亲和柱纯化目的蛋白,纯化产物进行酶活性的测定.结果:克隆获得的α-葡萄糖苷酶基因与NCBI收录的基因序列属等位基因,核苷酸位点有多处差异,氨基酸序列也有不同.构建表达载体并诱导表达后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,目标蛋白相对分子质量约为65kD,与理论值相符.目标蛋白量约占菌体总蛋白量的18%.同时,由纯化结果可以看出,以500mmol/L咪唑洗脱时的纯化效果最理想,蛋白纯度可达90%以上.对表达产物进行过酶活性初步检测证重组的阪崎肠杆菌α-葡萄糖苷酶能够分解4-硝基苯基-α-D-呋喃型葡萄糖,产生蓝绿色.结论:本研究中克隆获得了阪崎肠杆菌α-葡萄糖苷酶基因,通过构建pET22b( )-Glu表达质粒转化大肠杆菌可获得基因重组的高表达菌株,表达产物具有较好的酶活性,纯化后纯度可达90%以上.     

β-1,3- 葡聚糖酶基因克隆、表达及抗菌活性的研究

通过RT-PCR的方法分别从小麦和水稻的cDNA中克隆获得β-1,3-葡聚糖酶基因(Glu基因),分别命名为TaGlu9507和OsGlu30。它们的序列分析表明这两个克隆均含一个1002bp的开放阅读框(ORF),编码334个氨基酸,各自的N-端含有一个长20和29个氨基酸残基的信号肽序列。将不含信号肽序列的TaGlu9507和OsGlu30编码区DNA片段分别克隆进pET28-a(+)表达载体,并转入大肠杆菌BL21(DE3),经0.5mmol/L IPTG诱导3h后获得了高量表达,表达量分别占大肠杆菌可溶性蛋白的49.7%和26.7%,表达产物对黑曲霉、酵母等真菌生长均有较为明显的抑制作用。本结果更进一步表明β-1,3-葡聚糖酶基因是植物真菌病防治的潜在目的基因群之一。     

枯草芽孢杆菌精氨酸脱羧酶基因speA的表达与蛋白纯化

根据枯草芽孢杆菌（Bacillus subtilis）BJ3-2的精氨酸脱羧酶（ADC）的编码基因speA序列设计特异性酶切引物,克隆基因speA序列。测序结果显示,基因speA全长为1 473 bp,编码490个氨基酸,分子质量为58 ku。基因speA克隆至原核表达载体,获得重组菌pET28a-speA/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示,1.0 mmol/L的异丙基β-D-硫代半乳糖苷（IPTG）28 ℃诱导4 h,上清液和菌体均能表达出ADC蛋白,上清液经纯化、透析、冷冻干燥可获得纯度97%的ADC酶,酶联免疫吸附检测（ELISA）ADC酶活为16 780 U/mg。为speA基因的表达、纯化及酶学性质研究奠定了理论基础。     

Aspergillus ficuum内切菊粉酶基因在大肠杆菌中表达

研究内切菊粉酶基因在大肠杆菌中表达,利用基因工程的原理和方法,将来源于Asper-gillus ficuum JNSP5-06的内切菊粉酶基因(endoI)克隆到pET-28a(+),并在大肠杆菌BL21(DE3)中进行表达。重组菌经IPTG诱导后对表达产物进行酶活检测和酶水解产物分析。结果表明已成功构建表达载体pET28a-endoI,表达后的粗酶活为35 U/mL发酵液;重组酶水解菊粉的产物经TLC分析证实酶液具有内切菊粉酶活性,水解产物主要为低聚二糖、三糖和四糖。为其应用和工业化生产奠定基础。     

红曲菌Mn-SOD基因克隆、表达及序列分析

为得到产Mn-SOD工程菌及表达产物,通过设计出红曲菌H4000 Mn-SOD简并引物得到目的基因片段,再设计全长引物利用PCR技术扩增红曲霉Mn-SOD基因的全长序列。经EcoR Ⅰ和Hind Ⅲ酶切后连接至相同酶切的表达载体pET28a,并转化至E.coli BL21进行诱导表达。克隆得到的基因预测编码152个氨基酸,预测相对分子量为17 kDa。同时,将克隆得到的Mn-SOD基因与NCBI数据库进行比对,发现该序列与橙色红曲霉超氧化物歧化酶(SOD)基因相似度达到99%,与炭疽菌、米曲霉、黄曲霉的Mn-SOD基因也有较高的相似度。通过SDS-PAGE检测蛋白表达情况,目标蛋白相对分子质量约为19 kDa,与预测分子量基本相符。该表达蛋白在pH2.0保温处理1 h,仍具有较高的Mn-SOD酶活。     

烟草脉带花叶病毒基因组序列分析

为了探明烟草脉带花叶病毒危害和流行的机制,使用RT-PCR技术扩增获得TVBMV云南分离物YN9.1基因组全序列,并进行了基因组结构、序列同源性、氨基酸保守基序、系统进化和重组分析。结果表明:(1)YN9.1基因组具有Potyvirus属病毒典型特征,含有在Potyvirus属病毒中较为少见的NIb/CP切割位点Q/N;(2)YN9.1与其它分离物核苷酸序列同源性为90.5-91.1%,氨基酸序列同源性为95.2-96.2%。与YND核苷酸和氨基酸序列同源性最高;(3)对HC-Pro和CP保守基序进行了分析。YN9.1具有RITC、PTK、DAG等病毒蚜虫传播保守基序,其中RITC在Potyvirus属病毒中常见形式为KITC;(4)系统进化树分析结果显示,TVBMV进化形成2个组,云南分离物独立进化形成一组,TVBMV进化与地域具有明显的相关性;(5)重组分析发现PY为ZC1和YN的组内重组体,重组位点位于HC-Pro 3'末端和NIb 5'端。      

去盲肠公鸡对豆粕氨基酸标准消化率的比较研究

在比较分析无氮日粮法(NFD)和回归法(REG)测定去盲肠公鸡的内源氨基酸基础损失量(EAALb)的基础上,采用排空-强饲代谢试验测定豆粕的氨基酸(AA)标准消化率,为家禽饲料原料AA营养价值的评定提供理论依据。结果显示:(1)总AA的排泄量(mg/kg DMI)与摄入量(mg/kg DMI)间线性回归方程为Y=12 625.2+0.096X(R~2=0.759);(2)NFD法和REG法测定的EAALb分别为11 489.7、12625.2 mg/kg DMI,两者间差异不显著(P0.05);(3)豆粕总AA的表观消化率随日粮粗蛋白质(CP)水平的增加呈二次曲线变化规律,在CP12.0%时,达到相对恒定;经NFD和REG校正后得到的豆粕总AA标准消化率差异不显著(P0.05),且均不依赖于日粮CP水平,分别介于85.58%~89.61%和86.75%~90.05%。以上结果表明,NFD和REG均可用于测定EAAL_h,豆粕总AA和大部分AA的标准消化率不受日粮CP水平的影响,表明以豆粕AA标准消化率配制日粮具有较好的可加性。     

Cloning and expression of the N-acetylmuramidase gene from Streptomyces rutgersensis H-46

The N-acetylmuramidase SR1 gene from Streptomyces rutgersensis H-46 was cloned in Escherichia coli JM109 and expressed in E. coli BL21(DE3)pLysS. An open reading frame included the leader peptide region encoding a polypeptide of 65 amino acid residues and the mature SR1 enzyme region encoding a polypeptide of 209 amino acid residues. The overall G + C content of the mature enzyme gene was 67.6%, with 98.1% of G or C in the third position of the codons. The calculated molecular weight of the mature enzyme was 23,057 Da. The amino acid sequence of the mature enzyme showed a significant level of identity with bacteriolytic enzymes from Streptomyces globisporus (50.9% identity), Chalaropsis species (40.2% identity) and Saccharopolyspora erythraea (31.0% identity). The mature enzyme gene cloned into plasmid pET26b carrying a signal peptide, peIB, was expressed in E. coli BL21(DE3)pLysS. The signal peptide region was cleaved during the production of the enzyme. Specific activity of the enzyme purified from the transformant was almost identical to that of the native enzyme. Furthermore, the SR1 enzyme gene cloned with the leader peptide gene into plasmid pET28a was also expressed in E. coli. In this case, a proform-like protein was partially processed; 35 amino acid residues were cleaved but 30 amino acid residues remained. This proform like protein has approximately one-nineteenth the activity of the native enzyme. These results indicated that the native SR1 enzyme was produced in the following manner in the cells of S. rutgersensis H-46. The SR1 enzyme gene was translated to a pre-proform protein followed by the deletion of a signal peptide. Finally, the proform-like protein was processed by deletion of the remaining leader peptide.     

我国29种青稞的营养及功能组分分析

为了解我国不同青稞品种的营养及功能组分,对29种青稞的成分进行分析,包括碳水化合物、蛋白质、脂肪、氨基酸、灰分、水分及β-葡聚糖、总黄酮。研究结果表明:29种青稞中蛋白质含量变化范围为8.74%~13.16%,脂肪含量变化范围2.44%~4.48%,总氨基酸含量变化范围7.48%~11.86%,β-葡聚糖含量变化范围2.89%~6.11%,总黄酮含量变化范围0.11%~0.28%,灰分变化范围1.66%~2.95%,不同品种之间存在显著性差异;碳水合物和水分的变化范围分别为73.82%~78.30%和7.69%~9.68%,显著性差异不明显。29种青稞中均含有17种氨基酸,包括人体所需的8种必需氨基酸。其中,青稞XZDM00025的蛋白质、总氨基酸和总黄酮含量最高,青稞XZDM00027的β-葡聚糖含量最高,青稞XZDM00074脂肪含量最低。通过对比WHO/FAO理想蛋白质氨基酸模式,青稞的第1限制性氨基酸是甲硫氨酸,第2限制性氨基酸是赖氨酸,第3限制性氨基酸是异亮氨酸。采用聚类分析(组间连接-平方欧式距离)法将29种青稞分为7类,前4类都仅包括1个品种,第Ⅴ类包括6个品种,第Ⅵ类包括2个品种,第Ⅶ类包括17个品种,且各大类间营养和功能含量组分存在显著性差异。     

仿野生栽培的两种猴头菌营养成分分析与评价

以北京山区林下仿野生栽培的两种猴头菌(猴头菌H.erinaceus,HE5和珊瑚状猴头菌H.coralloides,SH2)子实体为材料,比较其多糖、三萜和矿质元素含量及氨基酸组成等,并采用国际通用标准对其蛋白质营养价值进行评价。结果表明:两种猴头菌子实体含有较高的多糖和三萜类物质,含量分别为6.29%~7.04%、1.84%~2.61%,并且这两类物质在SH2中的含量显著高于HE5。猴头菌HE5组成蛋白质的必需氨基酸总量与总氨基酸的比值为41.34%,必需氨基酸与非必需氨基酸的比值为70.49%,必需氨基酸指数(essential amino acid index,EAAI)为91.31,表明HE5为良好蛋白源。相比较来说,猴头菌SH2的EAAI为80.28,为可用蛋白源。并且,两种猴头菌均含有丰富的矿质元素,尤其是Zn、Fe和Ca含量较高。综上所述,猴头菌HE5达到理想蛋白源标准,具有较高的营养价值;珊瑚状猴头菌SH2含有较高的多糖和三萜等物质,表现出潜在的药用价值,有待于深入研究其关键成分和作用机理。     

Evaluation of the fixed nitrogen-to-protein (N:P) conversion factor (6.25) versus ingredient specific N:P conversion factors in feedstuffs

BACKGROUND: The crude protein (CP) of feedstuffs is important as an indicator of essential and non‐essential amino acids for livestock. The protein (P) level needs to be known accurately, to minimize the feeding of excess nitrogen (N) and to reduce N pollution. Laboratory methods for determining N content report N from amino acids, but also N from ammonia and from non‐amino acid sources. The determined CP based on 6.25 × N level typically overestimates the true protein of feedstuffs. RESULTS: Determined ingredient‐specific N:P conversion factors k_A, k_P and k were not equal to the standard 6.25 factor. The k_A had the highest value in all ingredients, which leads to the estimation of specific crude protein (SCP), which is closer to true protein (the summation of the total amino acid residues from amino acid analyses). The SCP(k_A) was lower than CP and true protein in all ingredients, demonstrating that CP might overestimate the actual protein in feedstuffs. CONCLUSION: Based on data from 677 feedstuff samples from 2009, it is concluded that the mean k_A should be 5.68 for corn, 5.64 for soybean meal, 5.74 for corn DDGS, 5.45 for poultry by‐product meal and 5.37 for meat and bone meal. Copyright © 2011 Society of Chemical Industry     

Nitrogen metabolism of early lactation cows fed diets with two different levels of protein and different amino acid profiles

Four multiparous Holstein cows (569+/-122 kg) surgically prepared with indwelling catheters in the mesenteric, portal, and hepatic veins and carotid artery were allocated in a 4 x 4 Latin square to determine the effects of dietary crude protein (CP) level and amino acid (AA) profile on N metabolism during early lactation (from 25 to 65 d in milk). Cows received their diets in two equal meals and were milked twice daily. The dietary treatments were: 18% CP with a high (18H) or a low (18L) quality AA profile, and 15% CP with a high (15H) or a low (15L) quality AA profile. The four diets were similar in net energy for lactation (1.75 NEL Mcal/kg) and contained the same amount of RUP (34% of CP). The quality of the AA profile pertained only to the essential AA (EAA), and was assessed by comparison with the EAA profile of casein and considered the potential contribution of EAA from ruminal bacteria. The 18H and 15H diets were supplemented with 50 and 25 g/d of ruminally protected Met, respectively. After 10 d on treatment, a blood flow marker (p-amino-hippurate) was infused into a mesenteric vein, and arterial, portal, hepatic, and mammary blood samples were obtained at 3, 6, and 12 h after feeding. Dry matter intake was similar across treatments (23.4+/-0.5 kg/d). Amino acid oxidation, and consequent urea production, in the liver were numerically greater with the 18% CP rations, and, as a result, arterial urea concentrations were greatest (P < 0.01) with these rations. The amount of total AA extracted by the mammary gland tended to be greater with the H than with the L diets (21.4 vs. 18.2 mmol/ h, respectively). Milk yield tended to be greater (P = 0.16) with the 18H and 15H diets (47.7 and 46.3 kg/d, respectively) compared with the 18L and 15L diets (45.9 and 44.6 kg/d, respectively). Also, milk CP and casein contents were greatest (P = 0.09) with the H diets compared with the L diets. Milk and plasma urea N were greatest (P < 0.01) with the 18% CP diets. The efficiency of N utilization for milk protein synthesis was greatest (P < 0.09) with the 15% CP diets. It is concluded that milk protein production during early lactation is less susceptible to variations in dietary CP contents than variations in the AA profile of the dietary protein.     

枯草芽孢杆菌QM11漆酶基因克隆表达及序列分析

为获得高效表达的产漆酶工程菌,将枯草芽孢杆菌（Bacillus subtilis）的漆酶（cotA）基因在大肠杆菌中表达。用PCR的方法从枯草芽孢杆菌（Bacillus subtilis）QM11基因组中扩增cotA基因,连接载体pET22b构建成表达载体pET22b-cotA,转化至E.coli BL21（DE3）进行诱导表达,最终通过SDS-PAGE检测蛋白表达情况。克隆得到的cotA基因含有1542个核苷酸,由513个氨基酸组成,预测相对分子量为58 ku,理论等电点为5.91,无信号肽,二级结构以β-折叠和无规卷曲为主,其中β-折叠占26.71%,无规卷曲占52.05%,与NCBI已公布的Bacillus subtilis漆酶cotA基因（Gen Bank:GQ184294.1）碱基序列有12个碱基的差异,其编码的氨基酸序列有4个发生了改变。通过SDS-PAGE检测,目标蛋白相对分子质量约为54 ku,与预测相对分子量基本相符。      

3个不同产地的松露氨基酸组成及营养价值评价

该试验测定了四川会东县、四川攀枝花以及云南永仁县松露中蛋白质、脂肪和氨基酸的含量,并对其蛋白质的营养价值进行系统评价。结果表明,3个产地的蛋白质量分数在15.19%~19.91%之间,脂肪含量在2.12%~2.53%之间。不同产地松露的总氨基酸含量存在差异,介于14.82%~17.25%之间,17种游离氨基酸中谷氨酸和天门冬氨酸含量较高,鲜味氨基酸和甜味氨基酸分别占游离氨基酸总量的37.37%和32.55%;EAA/TAA在32.10%~34.12%之间,EAA/NEAA在47.29%~51.84%之间。氨基酸比值系数分(SRC)均值为90.83,酪氨酸+苯丙氨酸的氨基酸比值系数(RC)最小,为第一限制性氨基酸。     

桑椹酒中氨基酸含量分析

建立采用氨基酸分析仪测定桑葚酒中18种氨基酸含量的分析方法。样品经氮吹去除乙醇处理后,采用酸/碱水解将其中的蛋白质和肽类化合物转化成游离氨基酸,采用氨基酸分析仪测定其含量。18种氨基酸在2.5～250.0 μmol/L的浓度范围内,峰面积和浓度呈良好的线性关系,相关系数R为0.998 8～0.999 9,检出限均＜0.50 mg/L,各化合物的加标回收率为72.8%～104.0%,重复性试验结果相对标准偏差（RSD）均＜5.0%。该方法可靠、准确、重现性好,为桑椹酒的营养价值评价提供参考资料。