Eureka lemon zinc finger protein ClDOF3.4 interacts with citrus yellow vein clearing virus coat protein to inhibit viral infection

Citrus yellow vein clearing virus(CYVCV)is a new citrus virus that has become an important factor restricting the development of China’s citrus industry,and the CYVCV coat protein(CP)is associated with viral pathogenicity.In this study,the Eureka lemon zinc finger protein(ZFP)ClDOF3.4 was shown to interact with CYVCV CP in vivo and in vitro.Transient expression of ClDOF3.4 in Eureka lemon induced the expression of salicylic acid(SA)-related and hypersensitive response marker genes,and triggered a reactive oxygen species burst,ion leakage necrosis,and the accumulation of free SA.Furthermore,the CYVCV titer in ClDOF3.4 transgenic Eureka lemon plants was approximately 69.4%that in control plants 6 mon after inoculation,with only mild leaf chlorotic spots observed in those transgenic plants.Taken together,the results indicate that ClDOF3.4 not only interacts with CP but also induces an immune response in Eureka lemon by inducing the SA pathways.This is the first report that ZFP is involved in the immune response of a citrus viral disease,which provides a basis for further study of the molecular mechanism of CYVCV infection.

苹果坏死花叶病毒CP基因原核表达及其抗血清制备

苹果坏死花叶病毒(Apple necrotic mosaic virus,ApNMV)是近年新发现的病原物,并且是与我国苹果花叶病症状高度相关的重要病毒,进行ApNMV外壳蛋白(coat protein,CP)基因原核表达、制备多克隆抗血清,以建立快速、灵敏、准确的ApNMV常规检测方法尤为必要.通过设计特异性引物,以RT-PCR方法成功获得ApNMV CP基因序列,插入到原核表达载体pET-28a(+)中构建重组质粒,转化至大肠杆菌BL21(DE3),利用IPTG进行重组蛋白(含His-tag)诱导表达.SDS-PAGE和Western blot分析结果表明,CP基因在大肠杆菌中获得了高效表达,用Ni Sepharose 6 Fast Flow进行蛋白纯化,回收共得到重组蛋白2.4 mg.在屏障环境下免疫2只SPF级新西兰兔,制备出多克隆抗血清,稀释400倍后仍能与ApNMV阳性苹果叶片样品发生免疫反应.由此证明,本研究建立的ApNMV间接ELISA检测方法具有较好的灵敏性,检测效率高,能够用于田间大量样品ApNMV的诊断.

中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用

中国小麦花叶病毒(CWMV)是浙江省农业科学院发现并鉴定的一种土传病毒,其RNA2编码的外壳蛋白(CP)在病毒侵染过程中发挥重要功能。为了检测该蛋白表达并分析其生物学功能,该研究采用RT-PCR方法从感染CWMV小麦病叶中扩增获得CP基因,并通过In-Fusion技术构建了CWMV CP重组表达载体,将其导入BL21(DE3)菌株诱导表达;原核表达的重组CP蛋白经镍柱亲和层析纯化后注射免疫家兔,收集抗血清经亲和层析纯化获得CP多克隆抗体。Dot-ELISA、ID-ELISA、Western blot显示,该抗体不仅对CWMV具有高度的特异性,而且其效价高达1∶2.048×10^(7)、灵敏度达6.25×10^(-2)ng。应用该抗体采用Dot-ELISA、Western blot方法检测田间小麦样品显示其可用于CWMV的准确诊断,这为CWMV病毒病检测、CP定量分析及其功能研究奠定了基础。

CP、Cys-C、RBP及其联合检测在喀什地区维吾尔族慢性肾脏疾病中的诊断价值

目的分析尿外泌体铜蓝蛋白(CP)、血清胱抑素C(Cys-C)、视黄醇结合蛋白(RBP)及其联合检测对喀什地区维吾尔族早期慢性肾脏疾病(CKD)的诊断价值。方法选取2019年1月—2023年1月在新疆喀什地区第一人民医院就诊的225例维吾尔族CKD患者纳入CKD组,另选取同期在体检中心接受健康检查的186例维吾尔族体检者纳入健康对照组。收集两组的尿外泌体CP、血清Cys-C及RBP值。采用logistic回归分析影响早期CKD的因素,绘制受试者工作特征(ROC)曲线分析CP、Cys-C、RBP及其联合检测对早期CKD的临床诊断效能。结果与健康对照组比较,CKD组患者的血清白蛋白、C反应蛋白、血尿素、血沉均明显升高(P<0.05),肾小球滤过率降低(P<0.05)。与健康对照组比较,CKD组尿外泌体CP、血清Cys-C及RBP表达均明显升高(P<0.001)。Logistic回归分析显示,尿外泌体CP、血清Cys-C及RBP是影响早期CKD的独立因素(P<0.001)。ROC曲线分析显示,与尿外泌体CP、血清Cys-C、血清RBP比较,联合检测(并联)诊断早期CKD的AUC、特异度均明显升高。结论早期CKD患者的尿外泌体CP、血清Cys-C、血清RBP均明显升高,尿外泌体CP、血清Cys-C、血清RBP均可用于诊断早期CKD,但联合检测(并联)的诊断效能更高,为尽早诊断CKD提供参考。

线虫surface coat proteins提取方法的建立与双向电泳分析

目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础。方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价。结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量。通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个。随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个。结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具。

Capillary Zone Electrophoretic Separation of Proteins in Polymer Coated Capillaries

Fused-silica capillaries used in capillary zone electrophoresis were statically coated with γ- glycidoxypropyltrimethoxysilane and epoxy polymer in order to suppress wall adsorption in the separation of proteins. It has been shown that a significant decrease in adsorption was obtained and eletroosmotic flow was the diminished in the pH range 3-5. However with higher pH values, appreciable peak deformation and decreases in the resolving power were observed. Under pH 5, the epoxy polymer coating was shown to be quite stable and exhibited reproducible separations from run-to-run and day-to-day over a period of time.

P1 of strawberry vein banding virus, a multilocalized protein, functions as a movement protein and interacts with the coat protein

Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.

李属坏死环斑病毒扬州分离物CP基因的原核表达及抗血清制备

李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)严重危害蔷薇科植物,在世界范围内普遍分布。为建立快速有效的PNRSV检测方法,依据PNRSV CP基因序列设计引物,并从桃叶片上扩增PNRSV-CP片段,将双酶切产物插入原核表达载体pET28a中,获得重组质粒pET28a-CP^(PNRSV),转化大肠埃希菌Rosetta菌株,IPTG诱导重组蛋白表达。SDS-PAGE电泳检测显示,在分子量约25 ku处有蛋白质条带,符合预期大小。镍柱亲和纯化PNRSV CP蛋白,以其纯化蛋白质免疫健康新西兰白兔,制备多克隆抗血清。Western blot、ELISA、Dot blot检测结果显示,制备的抗血清能检测到PNRSV CP基因在感病植物叶片中表达,而在健康植株中未检测到;制备的抗血清滴度稀释至1∶64000时,仍能与纯化蛋白质发生特异性反应。综上,制备的PNRSV抗血清特异性强、效价高,可用于PNRSV的快速检测。

Transformation of Coat Protein Encoding Gene from Soil-Borne Mosaic Virus into Wheat

CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat variety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 -5 mg L-1 Bialaphos were obtained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformation frequency had 0.99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstrated that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ to CP1- in T1 generation was 1.0 to 1. 3, and didn't agree with Mendelian rule. RT-PCR result from T2 plants showed that the alien gene CWMV-CP1 had stable expression in wheat genetic background.

Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus

Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.

Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in <i>Escherichia coli</i>and Product Identification by Mass Spectrometry

Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the SPLCV coat protein.

Transgenic Tobacco Lines Expressing Yam Mosaic Virus Coat Protein-Derived dsRNA Are Resistant to Yam Mosaic Virus

Yam mosaic virus (YMV), a Potyvirus, is a highly destructive pathogen of yam accounting for yield losses up to 40%. Apart from causing significant reduction in tuber size and quality, it restricts international exchange of germplasms. It thus becomes crucial to get resistant or at least virus-free planting materials for farmers. This study was aimed at inducing resistance to YMV in tobacco by RNA silencing. An RNAi construct containing 161 bp fragment of YMV-coat protein (CP) gene was developed and used to produce transgenic tobacco lines expressing YMV-coat protein (CP) derived double stranded RNA (dsRNA) via Agrobacterium-mediated transformation. Of the eight T1 transgenic lines inoculated with YMV, six (L1, L2, L3, L5, L7 and L8) showed immunity to YMV as no symptoms were detected, whereas two (L4 and L10) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and qPCR analyses of YMV-coat protein (CP). The presence of small interfering RNAs in transgenic lines after virus challenge indicates that the resistance was acquired through RNA silencing.

Application of M13 Phage Coat Proteins

M13 phage＇is a filamentous bacteriophage containing a circular single-stranded DNA molecular, which is surrounded by approximately 2 800 copies of protein PVIII. In addition, there are five copies of each of proteins PVII and PXI in one end, and five copies of each of proteins PVI and PUI on the other. These coat proteins have play an important role in the infection and assembly of M13 phage. With the development of phage display technology, these five coat proteins all can be used in phage display, which plays an increasingly important role in molecular detection and treatment.

地黄花叶病毒河南分离物的CP基因克隆及序列分析

为探究地黄花叶病毒(rehmannia mosaic virus,ReMV)在河南地黄上的发生与分布及其基因变异情况,从河南温县中草药园采集若干疑似受病毒侵染而呈现出花叶、枯萎等症状的地黄样品,用酚仿法提取地黄样品总RNA,通过PCR扩增目的基因地黄花叶病毒外壳蛋白(coat protein,CP)基因片段,经过回收纯化后,克隆至pMD19-T载体中,转化大肠杆菌DH5α,随机挑选4个阳性单克隆测序,最后将结果与NCBI数据库中已有的核苷酸序列进行比对,并结合NCBI数据库中已发布的不同地区ReMV分离物的CP基因序列,构建基于CP^(ReMV)核苷酸序列的ReMV不同地区分离物的系统进化树。结果表明:成功克隆出ReMV河南分离物的CP基因,将该序列上传至NCBI后获得GenBank登录号为OM964874;通过构建系统发育进化树,该分离物与郑州、山西、韩国报道的ReMV分离物亲缘关系较近,可能有相近传染源,而与美国、日本的分离物亲缘关系较远,存在一定的遗传分化,序列一致性分析结果与此一致。综上,这一研究扩大了ReMV群体的传播范围,丰富了其基因信息,为揭示ReMV的致病机制奠定了基础。

Genetic Characteristics of <i>Citrus Tristeza Virus</i>Isolates from Cultivated Citrus in China Based on Coat Protein Gene

Citrus tristeza virus (CTV) is an important citrus pathogen causing considerable economic loss to citrus production. Knowledge on genetic evolutionary of the CTV population in China remains limited. In this study, 1439 samples were collected from nine citrus-producing areas of China. The coat protein (CP) genes of CTV were amplified by RT-PCR, and sequenced to analyze the genetic evolution. Analysis of the base composition showed an AU preference pattern, with the GC content was lower than AU content. Nine CTV populations were clustered into one clade in neighbor-joining (NJ) tree, indicative of a close phylogenetic relationship among the populations in China. Analysis of molecular variation (AMOVA) revealed that 77.72% genetic variations of CTV populations were observed among populations, with an FST value of 0.223. The values of dN/dS and neutrality test of CP gene were ranged from 0.016 to 0.082 and -1.377 to 1.456, respectively, the results suggesting that all of nine CTV populations were relatively constantly maintained under purifying selection. Our study demonstrated the genetic characteristics and molecular evolution relationship of CTV populations in China, and provided a theoretical basis for scientific control of CTV.

Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

Capillary Zone Electrophoretic Separation of Basic Proteins with Coated Columns Prepared by Sol-Gel Technology

Coated capillary columns were prepared by sol-gel technology and used in the separation of basic proteins with capillary zone electrophoresis. The results indicated that a significant decrease in protein adsorption was obtained and EOF was also diminished to zero in the pH range of 3-10.

利用酵母双杂交系统筛选与柑橘黄化脉明病毒CP互作的寄主因子

【目的】柑橘黄化脉明病毒(citrus yellow vein clearing virus,CYVCV)是威胁我国柑橘产业稳定发展的主要病毒,但其在柑橘上的侵染和致病机制尚不清楚。本文以CYVCV的外壳蛋白(coat protein,CP)为诱饵筛选尤力克柠檬(Citrus limon Burm.f.)c DNA文库,利用生物信息学方法分析与其互作的寄主因子在病毒侵染和致病过程中可能发挥的作用。【方法】Trizol法提取尤力克柠檬叶片总RNA,用SMART法反转录合成First-Strand c DNA,以其为模板通过Long-Distance PCR获得ds c DNA,均一化处理后与线性化p GADT7质粒重组链接构建尤力克柠檬初级c DNA文库,重组质粒转化大肠杆菌DH10B获得尤力克柠檬大肠杆菌c DNA文库并对文库质量进行鉴定;PCR扩增CYVCV的CP序列并构建到载体p GBKT7上,鉴定诱饵质粒对酵母细胞的毒性以及CP蛋白对酵母报告基因的自激活性。将尤力克柠檬c DNA文库质粒转化含有诱饵质粒p GBKT7-CP的Y2H Gold酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His/X-α-Gal和SD/-Leu-Trp-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序比对获得候选基因,利用Uniprot在线网站的gene ontology(GO)注释候选基因,分析候选互作蛋白的生物学功能。根据分析结果,选取可能参与寄主抗病或症状发展的候选因子,扩增其CDS全长序列并构建到靶标载体p GADT7后分别与p GBKT7-CP共转化酵母细胞进行点对点酵母双杂交互作验证。【结果】尤力克柠檬大肠杆菌c DNA文库滴度为1.02×10^(8)cfu/m L,库容符合试验标准;成功构建酵母双杂交诱饵载体p GBKT7-CP,该载体没有自激活性且对酵母菌没有毒性;在SD/-Leu-Trp-His-Ade/X-α-Gal平板上筛选得到41个酵母阳性克隆,经序列相似性比对,去除重复,共筛得32个候选寄主因子;GO通路注释结果表明这些寄主因子参与多个叶绿体相关的生物过程,包括碳水化合物代谢、光合作用、光刺激反应、代谢分解和生物合成等过程;这32个寄主因子的分子功能多样,包括催化活性、水解酶活性、转移酶活性、蛋白质结合、转录因子活性和翻译因子活性等,其细胞组分涉及叶绿体、类囊体、膜、细胞质、细胞核和高尔基体等。从候选寄主因子中选取14个重要的蛋白与CP的一对一酵母双杂交验证结果表明,CP与这14个蛋白均发生互作。【结论】成功构建了尤力克柠檬c DNA文库,筛选出32个与CYVCV CP互作的尤力克柠檬寄主因子,分析重要的寄主蛋白功能,推测CYVCV CP通过与光系统Ⅱ放氧强化复合物组分蛋白(Psb P)、光系统I叶绿素结合蛋白(Lhca3)和1,5-二磷酸核酮糖羧化酶小亚基(Rbc S)等多个叶绿体相关蛋白的互作,影响光系统稳定、类囊体结构和叶绿素合成,从而导致光合作用降低以及叶绿体形态和功能受损,酵母点对点验证了CP与这些叶绿体相关因子的互作,这为揭示CYVCV CP参与病毒致病的分子机制提供了理论依据。

Hydrophilic Silica/Copolymer Nanoparticles and Protein-Resistance Coatings

Hydrophilic silica/copolymer nanoparticles of SiO2-g-P(PEGMA)-b-P(PEG) are prepared by silica surface-initiating atom transfer radical polymerization (SI-ATRP) of poly (ethylene glycol) methyl ether methacrylate (PEGMA) and poly(ethylene glycol) methacrylate (PEG), by using Three molar ratios of SiO2-Br/PEGMA/PEG as 1/42.46/19.44, 1/42.46/38.88 and 1/42.46/77.76. Their temperature sensitive behaviour, pH response and surface properties as protein-resistance coatings are characterized. 220 nm core-shell nanoparticles as P(PEGMA)-b-P(PEG) shell grafted on SiO2 core are formed in water solution, which gained LCST at 60。C - 77。C and good dispersion in water when pH > 5.0. The water-casted films by SiO2-g-P(PEGMA)-b-P(PEG) obtain a little rough surface (Ra = 26.8 - 29.7 nm). While, the introduction of P(PEG) segments could slight increase the protein-repelling adsorption of SiO2-g-P(PEGMA)-b-P(PEG) films (△f = ?6.96 Hz ~ ?7.25 Hz) compared with SiO2-g-P(PEGMA) films (△f = ?9.5 Hz). Therefore, SiO2-g-P(PEGMA)-b-P(PEG) could be used as protein-resistance coatings.

饲粮蛋白水平和酿酒酵母对秦川肉牛血常规指标和氮磷代谢的影响

【目的】研究不同蛋白水平饲粮及酿酒酵母(SC)对秦川肉牛血液生理生化指标和氮磷代谢的影响。【方法】选择18头14月龄初始体质量为(298±9.78)kg/头、健康的秦川肉牛(母牛)作为试验动物,采用2种蛋白水平(13.23%(A1)和(10.59%(A2))饲粮和3种酿酒酵母添加水平(0(B1),4×10^(8) CFU/kg(B2),8×10^(8) CFU/kg(B3))作为两因素进行饲喂试验。采用全收粪(尿)法测定各处理秦川肉牛粪便和尿液中的氮磷含量,试验结束时于晨饲前采用颈静脉穿刺法采血,测定血常规指标和酶活性,试验期55 d。【结果】①试验期11~55 d,饲粮中添加8×10^(8) CFU/kg SC显著提高了秦川肉牛的采食量(P<0.05),A1B3组秦川肉牛采食量最高,显著高于A1B1组(P<0.05)。②B3组秦川肉牛血清中甘油三酯含量显著低于B1组(P<0.05),A2B3组秦川肉牛血清中总蛋白、白蛋白、球蛋白、免疫球蛋白G和免疫球蛋白M含量均显著高于A2B1组(P<0.05)。③B3组秦川肉牛粗蛋白(CP)和中性洗涤纤维(NDF)的表观消化率较B1组分别显著提高了8.44%和16.90%(P<0.05),A2B3组秦川肉牛的粗蛋白、有机物和酸性洗涤纤维表观消化率最高。④与A2B1组相比,A2B3组秦川肉牛粪氮排放减少了31.28%,尿氮排放减少了19.67%,氮消化率提高了7.66%,氮沉积率提高了62.57%。⑤B2和B3组秦川肉牛的粪磷含量显著低于B1组,A2B3组秦川肉牛磷消化率、磷沉积率相较于A2B1组显著升高了35.96%和45.94%(P<0.05)。【结论】本试验条件下,添加8×10^(8) CFU/kg酿酒酵母能够提高秦川肉牛的采食量、免疫水平和营养成分消化率,减少氮磷排放。在10.59%粗蛋白水平下,饲粮添加8×10^(8) CFU/kg酿酒酵母饲喂秦川肉牛效果最好。