Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice （Oryza safiva L. ssp. japonica）

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.     

Studies on Preparation and Characteristics of Monoclonal AntibodiesAgainst Enrofloxacin and Cross-Reactivity of Related Fluoroquinolones

An ester activation method was employed to couple enrofloxacin(ENFX) to the carrierproteins BSA and OVA. The conjugates ENFX-BSA and ENFX-OVA were identified with an UVspectrophotometer and amino acid automation analysis instrument, and resulted in conjugateswith 48 ENFX molecules per carrier molecule(BSA). Splenocytes from mice immunized withENFX-BSA were fused with SP2/0 myeloma cells and hybridomas secreting antibodies againstenrofloxacin were selected and cloned. Two stable monoclonal antibodies, 2C5, 5D5 of thesubclass IgG2a, were isolated. Using antibody 5D5, an indirect competitive inhibitionenzyme-linked immunosorbent assay (Ci-ELISA) was developed for the quantitative detectionof enrofloxacin and its metabolites. The IC50 of the standard curve was 21.67 ng mL^(-1) andthe limit of detection for enrofloxacin was 0.13 ng mL^(-1). This method was sensitive and hada linear range from 0.13 to I0 000ngmL^(-1) (r=-0.9782). Monoclonal antibody 5D5 exhibitedhigh relative affinity to enrofloxacin, and the cross-reactivities with ciprofloxacin,marbofloxacin, sarafloxacin and danorfloxacin were 110.8, 27.40, 71.05 and 37.41%,respectively. Three non-fluoroquinolones of cefadroxil, chloramphenicol, sulfadimethoxinewere tested and there was no cross-reaction between them.     

Impact of Long-Term Atrazine Use on Groundwater Safety in Jilin Province,China

The long-lasting application of representative herbicide atrazine (ATR) has given rise to the accumulation of its residues in the groundwater. To investigate the impact of long-term ATR use on groundwater safety, the residues of ATR and its metabolites, desethylatrazine (DEA), deisopropylatrazine (DIA) and hydroxyatrazine (HA) were monitored in groundwater and top soil at the major corn growing region of Qian’an and Gongzhuling in Jilin Province, China. The residues of the target compounds were analyzed by UPLC-MS/MS. The limits of detection (LODs) of ATR, DEA, DIA, and HA were 0.5, 0.5, 5, and 0.5 ng L-1 in groundwater and 0.33, 0.33, 3.3, and 0.33 μg kg-1 in soil. The target compounds were found in 94% of groundwater samples and 100% of soil samples. The compounds detected most frequently in groundwater were ATR (89%), DEA (64%) and HA (17%), whereas in soil were ATR (97%), DEA (36%) and HA (97%). DIA was not detected in any determined groundwater and soil sample. Average residues were 106.8 ng L-1 for ATR, 0.9 ng L-1 for DEA and 0.3 ng L-1 for HA in groundwater, whereas 11.1 μg kg-1 for ATR, 0.4 μg kg-1 for DEA and 7.8 μg kg-1 for HA in soil. ATR residues detected in groundwater samples were below standards for drinking water quality (GB5749-2006, 2 μg L-1), while the total residues of ATR and its chloro-s-triazine metabolites (DEA and DIA) were below current WHO (World Health Organization) guideline value (GV, 0.1 mg L-1). In addition, concentrations of HA in groundwater were determined below current WHO GV (0.2 mg L-1). The results indicated that ATR is safe to be used in Jilin Province under the current application scheme. However, total residues of ATR and DEA were detected in nearly all wells, thus, it is necessary to pay attention on groundwater monitoring for ATR and its metabolites.     

Detection of GLV, OYDV and LYSV in potato onion （Allium cepa L., Aggregatum group） by RT-PCR

Three pairs of primers were designed and synthesized from nucleotide sequences of garlic latent virus (GLV), onion yellow dwarf virus (OYDV), and leek yellow stripe virus (LYSV) by using PCR primer design software. The expected fragments about 170 bp, 287 bp, and 191 bp were amplified by RT-PCR for GLV, OYDV, and LYSV, respectively in disease-infected plants of potato onion (Allium cepa L., Aggregatum group), but such fragments were not obtained from healthy-looking plants and virus-free seedlings of shoot-tips. The amplified products of GLV, OYDV and LYSV were cloned into pGEM-T vectors, and transformed into Escherichia coli. JM109. The recombinant plasmids were obtained and sequenced. The nucleotide sequences were compared with corresponding viral nucleotide sequences reported in GenBank by performing a NCBI BLAST. The analysis showed that their homology attained 75% to 90%,89.5% to 96.1%,and 91.6% to 96.3% in GLV, OYDV, and LYSV, respectively. The total RNA of 6.34 μg·μL~(-1) from infected plants was diluted to a series of 10~(-1) to 10~(-5) and the detection sensitivity of RT-PCR was 10~(-4) (about 4 ng). Thus, a method of identification and detection by RT-PCR of GLV, OYDV, and SLYV was established.     

Studies on Single Cell Culture in vitro in Wheat--The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742),the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995.The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11.53-17.70%).Several types of variation were found in the studies,espercially the type with higher protein content than that of cultivar NE 7742(non-culture parent).Among them.-20.69% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential.The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE7742,the content of gliadin increased in some somaclones and decreased in others.However,the percentages both globulin and glutenin tended to increase.The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase.the variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro.The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly by glutenin.The results mentioned above bemonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.     

Production and Characterization of Polyclonal Antibody for the N- Methylcarbamate Insecticide Metolcarb

The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.     

Establishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie in Chinese Short-Tailed Han Sheep by Monoclonal Antibody

The method of immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep was established using monoclonal antibody. Genomic DNA was isolated from Chinese Short-tailed Hart sheep blood. Using the polymerase chain reaction technique, PrP27-30 gene sequence was amplified from Chinese Short-tailed Han sheep genomic DNA. By recombinant DNA technology, the recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained. Then, using standard methodology of myeloma cell fusion, a panel of monoclonal antibodies was generated. With mAbs, scrapie in Chinese Short-tailed Han sheep was detected by immunohistochemistry assay. The recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained and a panel of six hybridoma cell lines secreting specific antibodies to Chinese Short-tailed Han sheep PrP27-30 related to scrapie was obtained with one fusion between myeloma Sp2/0 and spleen ceils from mice immunized with the purified recombinant protein. Four hybridoma cell lines can be used in immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep. So that the special monoclonal antibody developed in author＇s institute can be used to detect PrP^sc of scrapie in Chinese Short-tailed Han sheep by immunohistochemistry in China.     

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.     

Application of Molecular Markers Linking to Cytoplasmic Male Sterile Loci to Assist Maintainer Line Selection and Their Selection Efficiency in Welsh Onion （Allium fistulosum L.）

Cytoplasmic male sterility exists widely in most natural populations of welsh onion （Alliumfistulosum L.）, which makes it possible to breed out many male sterile lines for heterosis utilization. Unfortunately, the breeding of cytoplasmic male sterility in welsh onion has a little progress due to the limitation of its biological characteristic and traditional selection approach. To study the feasibility and the efficiency of utilizing marker assisted selection for male sterile lines in welsh onion, one SCAR marker, SCS13, and one RAPD marker, S2002400, which could distinguish between N and S cytoplasm in several welsh onion cultivars, were identified. The two markers were then confirmed by Southern blotting, and used to screen the N or S cytoplasm of individual plants in seven welsh onion cultivars in this study. Male sterile and fertile plants were evaluated by aceto-carmine dying. The frequency of N-cytoplasmic plants and maintainer genotype was calculated in the seven open populations of welsh onion. The minimum number of plants needed to identify a maintainer was evaluated to be 95% reliable. Results showed that 20 to 80% decrease of crosses and self-crosses for identifying a maintainer genotype could be achieved by the marker-assisted selection compared with traditional selection method. It was proved that the molecular markers could precisely identify cytoplasmic types individually, performed by one generation of cross and two generations of testcrosses and self-crosses. Finally, several maintainer genotype plants were selected with the help of the two markers in the seven cultivars. The screened markers could assist and accelerate sterile and maintainer lines selection with less labor and cost.     

莱克多巴胺人工免疫原合成及抗体特性

将莱克多巴胺(RAC)进行化学修饰引入羧基活性基团,合成具有半抗原结构特征的RAC-戊二酸酐半醛化合物(RAC-SA);采用混合酸酐法(MA)将RAC-SA与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成人工免疫原BSA-RAC和包被抗原OVA-RAC,用红外(IR)、紫外(UV)和凝胶电泳(SDS-PAGE)进行鉴定,推算分子结合比;用BSA-RAC免疫新西兰白兔,间接ELISA测定多克隆抗体(pAb)效价,阻断ELISA鉴定其敏感性,琼脂双扩散试验、WestGold膜杂交试验和交叉反应试验鉴定其特异性。结果表明,BSA-RAC偶联成功,分子结合比为24.5∶1;获得了高价、敏感、特异的RAC pAb,为RAC残留免疫学检测方法的建立奠定了基础。     

抗沙丁胺醇单克隆抗体制备与鉴定

[目的]制备特异性抗沙丁胺醇单克隆抗体,为其免疫学检测方法的建立奠定基础。[方法]用SAL-BSA抗原免疫BALB/c小鼠;使用细胞融合技术建立抗SAL的单克隆抗体（SAL mAb）杂交瘤细胞株;体内诱生腹水法制备SAL mAb,并鉴定其免疫学特性。[结果]筛选出2D9和1C4两株杂交瘤细胞,其细胞培养上清效价达1∶104,腹水效价达1∶106;免疫球蛋白亚型鉴定为IgG1;抗体对SAL的半数抑制浓度（IC50）为1.14 ng/ml;与沙丁胺醇和盐酸克伦特罗的交叉反应率（CR）分别为100.00%和26.09%,与其他化合物无交叉反应。[结论]获得了高效价、敏感且异性的抗SAL抗体,为沙丁胺醇残留的免疫学检测方法的建立奠定了基础。     

恩诺沙星单克隆抗体杂交瘤细胞株的建立及其免疫学特性

将恩诺沙星(ENR)偶联于载体蛋白BSA,形成完全抗原(BSA-ENR)并免疫BALB/c小鼠,应用细胞融合技术制备分泌抗恩诺沙星的杂交瘤细胞株,用体内诱生腹水法生产ENR单克隆抗体。结果筛选出4G1-B3,4G1-G1和4G1-B9 3株敏感特异的杂交瘤。3株单抗对ENR的半数抑制浓度(IC50)分别为1.04 ng/mL,2.44 ng/mL,4.24 ng/mL。4G1-B3与环丙沙星有0.02%的交叉反应,与其他竞争物的交叉反应率均小于0.01%;4G1-G1和4G1-B9与其他竞争物的交叉反应率均小于0.01%。     

蛋白质纳米铁共振光散射探针的研究

[目的]探索定量测定核酸和蛋白质等生物大分子的方法。[方法]采用Fe(OH)3纳米粒子作为共振光散射探针,研究纳米铁-BSA反应体系测定生物大分子的最优条件及效果。[结果]纳米铁-BSA体系的最佳反应条件是pH值为7.4,纳米铁溶液的加入量为0.5ml,定量测定时选择吸收波长和发射波长均为477 nm。将配好的溶液放置35 min后再进行测定稳定性较好。在上述优化条件下,用该体系测定BSA的线性范围在0.07~0.60μg/ml,线性方程为IRLS=38.0+1583.3C(μg/ml),相关系数为0.99712,检出限为10.2 ng/ml。控制误差在5%以内,该体系对较高浓度的金属离子以及从1.0~100.0μg/ml浓度范围内的氨基酸有很强的抗干扰能力。[结论]该方法简便、快速、可靠且灵敏度高,可用于实际样品的测定。     

抗庆大霉素单克隆抗体的制备及其初步应用#br#

 【目的】制备抗庆大霉素(gentamicin,GM)的高亲和力特异性单克隆抗体,并鉴定其免疫学特性,为进一步研究GM快速检测试剂盒和试纸条打下基础。【方法】用EDC法将BSA和OVA分别和GM偶联作为免疫原或包被原,并经聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定。用合成的BSA-GM免疫Balb/c小鼠,经过4次免疫后,用间接ELISA和阻断ELISA选择细胞融合备用鼠,选择高效价、敏感的小鼠进行抗原超强免疫;取其脾细胞应用杂交瘤技术与骨髓瘤细胞建立分泌GM 单克隆抗体(monoclonal antibody,mAb)的杂交瘤细胞株;用体内诱生腹水法制备GM mAb,对GM mAb的效价、敏感性和特异性等免疫学特性进行鉴定,;应用阻断ELISA试验原理组装GM-Kit,并对鲜奶中添加的GM标准样品进行测定。【结果】SDS-PAGE电泳鉴定表明BSA-GM人工抗原偶联成功;免疫的3只小鼠血清抗体效价均达到10-3;其中2号小鼠血清GM抑制效价较高且IC50最低,达17.28 ng•mL-1,融合后筛选出5E2-D7、1A3-A9、5E2-A12和2A6-B5共6株敏感特异的杂交瘤细胞,其细胞培养上清液效价分别为1﹕1600、1﹕800、1﹕800和1﹕800,腹水效价分别为1﹕2.05×107、1﹕5.12×106、1﹕2.56×106和1﹕2.56×106,5E2-D7株对GM的IC50为0.30 ng•mL-1,与链霉素、卡那霉素等其他氨基糖苷类抗生素无交叉反应性;检测鲜牛奶样的平均添加回收率分别为96.0%,平均变异系数均低于15%。【结论】本试验获得了高效价、敏感、特异的抗GM mAb,为GM残留检测奠定了坚实的基础。     

盐酸克伦特罗单克隆抗体的制备及其特性

以戊二醛法将盐酸克伦特罗 (Clenbuterol,CL)连接到牛血清白蛋白 (BSA)上制备抗原(BSA -CL) ,并以BSA -CL免疫Balb/c小鼠 ,应用杂交瘤技术将免疫鼠脾细胞与NS0细胞融合 ,建立分泌CL单克隆抗体的杂交瘤细胞株。通过对杂交瘤细胞培养上清的检测、鉴定 ,筛选出 1 7株高亲和力的杂交瘤细胞株 ,其中C - 4C9、C - 2D6、C - 4G1和C - 2H6的培养上清ELISA效价为 1∶5 1 2 ,1∶640 ,1∶81 0和 1∶2 5 6;腹水ELISA效价为 5× 1 0 - 6,1 .7× 1 0 - 5,2×1 0 - 6和 3.3× 1 0 - 5。 4株单抗与 β -肾上腺素激动剂沙丁胺醇的交叉反应性为 0 .79% ,与肾上腺素、去甲肾上腺素、异丙肾上腺素及畜禽常用抗生素无交叉反应性。可以作为制备CL快速检测的特异性单克隆抗体应用     

莱克多巴胺单克隆抗体的制备及特性鉴定

莱克多巴胺与牛血清白蛋白偶联,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞杂交,获得1株能稳定分泌莱克多巴胺单克隆抗体的杂交瘤细胞株,制备腹水,间接ELISA方法测定腹水抗体效价为1∶30 000。该抗体与莱克多巴胺的交叉反应率为100%,与克仑特罗的交叉反应率为0.01%。该单克隆抗体可用于对动物肉品或尿液中莱克多巴胺残留检测试剂盒或试纸条的开发。     

克伦特罗残留GC-MS检测方法的建立及对自制试纸条性能的确证

建立了猪尿液中盐酸克伦特罗(CL)残留的GC-MS检测方法,并对自主研制的CL残留快速检测免疫金标试纸条(CL-Strip)进行了比较分析.结果表明,GC-MS的检测限为0.5ng/ml,CL-Strip的检测限为1.0ng/ml:CL-Strip与GC-MS两者的加标测定实验结果完全一致;用CL-Strip检测出的100份阴性尿样和18份阳性尿样,与GC-MS检测结果完全一致.说明CL-Strip与GC-MS同样灵敏、准确、稳定,由于CL-Strip具有简便、直观、快速、敏感、特异、准确等优点,建议在CL残留快速检测中推广应用.     

莱克多巴胺单克隆抗体的研制

莱克多巴胺(Ractopamine)与戊二酸酐、氯甲酸异丁酯反应后,与牛血清白蛋白偶联,免疫BALB/c小鼠,用杂交瘤技术获得2株稳定分泌抗莱克多巴胺单克隆抗体的杂交瘤细胞株,并制备腹水,间接ELISA方法测定腹水抗体效价为1∶3.2×107。该抗体与克仑特罗交叉反应率为0.3%,体外传代培养和冻存复苏后抗体分泌稳定。获得的单克隆抗体可用于肉品中莱克多巴胺残留检测和莱克多巴胺残留检测试剂盒的开发。     

莫能菌素人工抗原合成及其多克隆抗体的制备

通过莫能菌素羟基与琥珀酸酐反应,合成半抗原莫能菌素-琥珀酰半酯,采用混合酸酐法将其与载体蛋白BSA偶联制备人工抗原,以此人工抗原免疫新西兰大白兔获得多克隆抗体.试验结果表明,莫能菌素结合抗原免疫家兔制备的抗体对莫能菌素具有很高的特异性、亲合性和选择性.用此抗体建立的莫能菌素间接竞争酶联免疫吸附检测方法的标准工作曲线I50值为37.9 ng/ml,最低检测限(I10)为2.09 ng/ml.