禽副粘病毒2型(PMV-2)群特异性单克隆抗体的研究

禽副粘病毒2型（Yucaipa株）鸡胚成纤维细胞适应毒经差速离心和蔗糖密度梯度离心法提纯，免疫Balb/c小鼠，取脾细胞与SP2/0骨髓瘤细胞融合，经间接ELISA方法筛选，有限稀释3次克隆，获得了两株分泌PMV-2单克隆抗体的杂交瘤细胞株6E9和7E5。经鉴定两株单抗均为IgG1亚类，杂交瘤细胞的平均染色体分别为96条和98条。细胞培养上清及腹水效价分别为1：25600、1：25600、1：51200和1：10^5、1:10^7。6E9和7E5单抗能稳定分泌抗体且与NDV、AIV、EDSV无交叉反应。间接ELISA实验表明4株PMV-2（Yucaipa株、F4株、F6株和F8株）均含有单抗6E9和7E5所针对的位点。     

单克隆抗体捕捉猪瘟病毒抗原ELISA方法的建立

分别用原核表达的猪瘟病毒（CSFV）主要抗原E2蛋白和猪瘟基因疫苗免疫BALB/c小鼠,通过细胞融合与克隆筛选出5株稳定分泌CSFV抗体的杂交瘤细胞株1E2、1G7、3A2、3B7和4B6.间接免疫荧光和Western-blotting试验结果表明,5株单抗均与CSFV E2蛋白和全病毒抗原反应.将筛选的CSFV特异性单抗1E2、3B7和4B6纯化后等量混合后包被酶标板（捕捉抗体）,与兔抗CSFV IgG（检测抗体）联合应用,建立起CSFV抗原捕捉ELISA（AC-ELISA）方法.随后采用方阵滴定法确定了单抗与多抗的最适工作浓度及判定检测结果的OD450临界值.最后以建立的AC-ELISA检测CSFV细胞培养物、CSFV攻毒死亡猪的病料和临床猪瘟组织样品,结果表明,该方法敏感、特异、重复性好,与病毒分离和RT-PCR方法符合率分别为86.2%和90.3%.     

IBDV H1株VP2基因的原核表达及其产物的复性

应用RT-PCR技术从传染性腔上囊病病鸡总RNA中克隆出1461bp的VP2基因，将其克隆于pET-28a载体，筛选并构建了pVP2表达载体。经IPTG诱导，在其宿主菌BL21（DE3）中成功表达了53．7ku的蛋白，SDS-PAGE和Western-blotting分析结果显示，VP2表达产物以包涵体形式存在，可与鸡IBDV抗血清及1株IBDV单抗发生特异性反应。将VP2表达产物进行纯化和复性，用复性前后的蛋白分别与特异性多抗进行Dot-ELISA检测，结果，复性后蛋白的反应活性比复性前增强了10倍；用复性后的蛋白与IBDV单抗进行Dot-ELISA，结果显示，VP2蛋白与单抗1H4、1E12、583、EA6、3c7反应强（＋＋＋）；与4E4、1H11反应中度（＋＋）；与4E5、3C4、1E11、3H9反应较弱；而与EC6、3D12、1A1无反应。     

场间种猪群猪瘟E2蛋白灭活疫苗与猪瘟活疫苗免疫效果对比

为准确了解、比对不同种猪场猪群免疫猪瘟E2灭活疫苗与猪瘟C株 （脾淋源、ST传代细胞源）活疫苗效果。对A、B、C、D、E、F 6家猪场1 672头母猪免疫对应猪瘟疫苗后,分别采集6家猪场的20头、30头、10头、20头、10头、10头共计100头母猪的血清样品进行间接ELISA检测。结果显示100份样品中猪瘟抗体阳性率100%;其中免疫猪瘟C株 （脾淋源、ST传代细胞源）活疫苗组A、B、C场抗体效价OD均值分别为2.0662、2.0029、1.8147,离散度分别为41.86%、28.89%、43.85%;免疫猪瘟E2灭活疫苗D、E、F场抗体效价OD均值分别为2.6587、2.2537、2.4862,离散度分别为21.75%、7.72%、9.76%;经两样本均数比较T检验分析,2个试验组内抗体效价OD值差异不显著,2个试验组间抗体效价OD值差异极显著。     

父母代蛋鸡在饲料中添加沙棘果渣对种蛋孵化的影响

为研究饲料中添加沙棘果渣对种蛋孵化的影响，选取30 w和体重相近且健康的海兰褐种蛋鸡600只，随机分成4组，各组设3个重复，每重复50只。对照组（CT组）饲喂基础日粮，试验组（E1、E2、E3组）分别在基础日粮中添加不同比例（2%、4%、6%）的沙棘果渣。饲喂5周，第35周结束后，采集精液人工授精。在授精后的第4天，各重复随机抽取200枚种蛋进行孵化，每组600枚，总计2400枚。结果表明：（1）试验组E1、试验组E2和试验组E3受精率分别较对照组提高1.41%、2.84%、5.14%(P <0.05)。（2）试验组E1、试验组E2和试验组E3受精蛋孵化率分别较对照组提高1.57%、2.93%、4.98%(P <0.05)。试验组E1、试验组E2和试验组E3入孵蛋孵化率分别较对照组提高3.02%、5.85%、10.38%(P <0.05)。（3）试验组E1、试验组E2和试验组E3健雏率分别较对照组提高1.18%、2.14%、5.28%(P <0.05)。（4）试验组E1、试验组E2和试验组E3平均初生重分别较对照组提高6.73%、14.08%、21.47%(P ...     

3株单抗对新城疫病毒HN抗原变异的分析

以新城疫病毒(NDV)LaSota株为免疫原研制了3株针对NDVHN蛋白的单抗,分别命名为1E5、C3-B7和E6-F11。3株单抗与NDV不同毒株在血凝抑制试验(HI)、ELISA和病毒中和试验中的反应性不同,而同一株单抗与同一个NDV毒株在HI、ELISA以及病毒中和试验中的反应性一致。其中,单抗1E5与NDV标准株(LaSota、F48E8)、8个NDV分离株反应阳性,而与9个NDV分离株反应阴性;单抗C3-B7、E6-F11与所有NDV毒株反应均为阳性。结果表明,3株单抗均是针对HN蛋白上的中和位点,国内部分NDV流行株的HN抗原至少有1个抗原位点发生了变异。     

养鸡补充维生素的12种情况

（1）在鸡患脂肪肝综合征时,要多添加5%的氯化胆碱和10%的多维素。 （2）当鸡群有应激反应（接种疫苗、转群等）时,应多添加10%的维生素A、维生素E、维生素D和维生素C。 （3）当鸡群有慢性病（慢性呼吸道疾病）时,也应多添加10%的维生素A、维生素E和维生素C。     

鸭星状病毒单克隆抗体的制备及鉴定

为了制备针对鸭星状病毒(Duck astrovirus,DAstV)衣壳蛋白的单克隆抗体,以DAstV C-NGB株为免疫原,用ORF2重组蛋白作为抗原进行筛选,获得7株稳定分泌抗DAstV ORF2蛋白单抗的杂交瘤细胞。取E5C1株和C10F6株进行鉴定,结果表明,2株单抗均为IgM,轻链均为κ型;细胞上清和小鼠腹水的效价分别为10~5和10~6以上;免疫印迹试验表明,2株单抗均能识别重组ORF2蛋白;特异性试验表明,E5C1株杂交瘤细胞的培养上清只与DAstV反应。     

抗猪圆环病毒Ⅱ型ORF2蛋白单克隆抗体的制备及鉴定

利用本室构建的质粒PGEX-ORF2特化BL21（DE3）．经IPTG诱导表达．得到PCV2-ORF2的重组蛋白，经复性纯化后作为免疫原．采取常规免疫与体外脾脏免疫结合的方式免疫BALB／c小鼠，取其脾细胞与Sp2／0的骨髓瘤细胞融合．经间接酶联免疫吸附试验（ELISA）筛选，获得了2株抗PCV2-ORF2蛋白的单克隆抗体的杂交瘤细胞．分别命名为382F4和9C3132，其细胞培养上清效价分别为1：1024、1：512，小鼠暖水效价分别为1：204800、1：102400。ELISA结果显示，382F4和9C3D2仅与融合表达的PCV2-ORF2蛋白反应，而与PGEX-KG表达的蛋白、猪细小病毒（PPV）、猪繁殖与呼吸综合征病毒（PRRSV）不反应．说明此2株单抗特异性好。间接免疫荧光结果显示．382F4和9C3D2能与PCV2发生反应，而不与PCV1发生交叉反应．表明所获得的2株单抗是PCV2型特异性的。这2株单抗的获得为进一步研究PCV2-ORF2基因的功能．及建立准确快速的鉴别PCV1和PCV2的诊断方法提供了强有力的手段。     

不同剂量包虫病基因工程亚单位疫苗EG95免疫牦牛后的抗体水平测定

为比较和评价不同剂量包虫病基因工程亚单位疫苗EG95免疫牦牛后的抗体差异,随机选择1岁左右的牦牛50头,平均分成A、B、C、D、E 5组,分别按照1、2、3、4、5头份剂量（每头份含EG95 50 μg）进行一免和二免（间隔28 d）;免疫前及二免后15 d采血,应用间接ELISA法检测血清中的特异性抗体。结果显示：二免后A、B、C组的抗体阳性率分别为30%、40%、50%,与E组的90%存在显著性差异（P＜0.05）,D组阳性率为80%,与E组无显著性差异（P＞0.05）;A组和B组抗体合格率均为0,C、D、E组的抗体合格率分别为10%、40%、70%,E组抗体合格率明显高于其他组（P＜0.05）。试验结果表明,采用EG95基因工程亚单位疫苗按5头份剂量免疫,间隔28 d后二免,对牦牛的免疫效果较为理想。     

补体介导的单克隆抗体对细粒棘球蚴原头蚴杀伤作用的初步体外研究

补体对体外细粒棘球蚴原头蚴有较强杀伤作用，单抗介入后能加强这一作用。由此表明宿主体液因子对原头蚴的免疫机理可能有3，即补体经典途径、补体旁路途径和抗体直接作用。     

马立克氏病病毒抗独特型单克隆抗体的研究

应用纯化的D_(11)~4株抗马立克氏病病毒（MDV）中和性单克隆抗体（McAb_1）,以SPA-IgG法和脂质体-IgG法制成免疫原,分别免疫BALB/c小鼠,应用B淋巴细胞杂交瘤技术进行细胞融合。以两步ELISA筛选法获得了3株抗独特型单克隆抗体（McAb_2）,分别命名为1B_8、10C_1和3D_5,其Ig亚类分别为IgG_1、IgG_1和IgG_(2b),染色体数在92～102之间。在捕获阻断ELISA中,McAb_2在1:20～1:80000稀释可不同程度地阻断MDV抗原与McAb_1的结合,阻断率为2.66％～76.40％。用纯化的McAb_2制成油佐剂苗,免疫20日龄雏鸡,经3次免疫后10d采取血清,可测得不同滴度的针对MDV抗原的抗体。这提示3株McAb_2的可变区构型和与McAb_1结合的MDV抗原决定簇或其邻近结构相似,有可能用于MD抗独特型疫苗的研究。     

细粒棘球绦虫BAG3和EB1基因的克隆、表达及其在原头蚴细胞凋亡中的作用

旨在探究细粒棘球绦虫BAG3(Eg-BAG3)和EB1(Eg-EB1)的分子特征及在原头蚴细胞凋亡过程中的作用,采用原核表达方法获得重组Eg-BAG3和Eg-EB1,利用生物信息学、蛋白免疫印迹及免疫荧光定位方法对Eg-BAG3和Eg-EB1的分子特征进行了初步探究,随后利用10 mmol·L^-1H₂O₂诱导原头蚴细胞凋亡,qRT-PCR方法检测Eg-BAG3和Eg-EB1的转录水平,并通过RNA干扰技术进一步探究二者与原头蚴细胞凋亡之间的关系。结果显示,Eg-BAG3含有BAG蛋白家族特征性的BAG结构域,Eg-EB1含有内吞蛋白家族特征性的BAR结构域,二者分别属于典型的BAG蛋白和内吞蛋白。免疫荧光定位结果显示Eg-BAG3广泛分布于细粒棘球绦虫的各个发育时期,Eg-EB1主要定位于原头蚴皮层、吸盘及顶突和包囊生发层,而在成虫未见特异性分布。H₂O₂可以成功诱导原头蚴细胞凋亡,且原头蚴中Eg-BAG3和Eg-EB1的相对转录水平都随着H₂O₂处理时间的延长而显著上升,在8 h后的相对转录水平与0 h相比具有显著性差异(P<0.05)。RNA干扰结果显示,Eg-BAG3干扰组原头蚴细胞凋亡率显著高于未干扰组(P<0.05),而Eg-EB1干扰组原头蚴细胞凋亡率低于未干扰组(P<0.05),表明Eg-BAG3在H₂O₂诱导的原头蚴细胞凋亡过程中起到抗凋亡作用,而Eg-EB1起到促凋亡作用。Eg-BAG3可以抑制氧化应激诱导的原头蚴细胞凋亡而Eg-EB1促进细胞凋亡,且二者可能参与调控不育囊的形成。     

吉氏巴贝斯虫单克隆抗体制备的研究

应用淋巴细胞杂交瘤技术制备杂交瘤细胞株 ,经间接萤光抗体法筛选和克隆 ,获得了 5株能稳定分泌吉氏巴贝斯虫特异性抗体的杂交瘤细胞株 ,分别命名为C3B5、M8B7、E9C5、G6 D8、H2 A7。经过鉴定 ,这 5株杂交瘤细胞分泌的单克隆抗体亚类及相对分子质量分别为IgG2b,1 8× 1 0 4 ;IgG2a,1 8× 1 0 4 ;IgG1 ,1 8× 1 0 4 ;IgM ,3 2× 1 0 4 ;IgM ,1 8× 1 0 4 。腹水效价为 1∶1 0 4 ～ 1∶1 0 5。其中E9C5杂交瘤细胞株分泌的单克隆抗体是一种保护性抗体 ,经对实验感染吉氏巴贝斯虫小鼠体内虫体的杀虫试验证明具有较强的杀灭作用。     

Effects on chick growth of adding various non-protein nitrogen sources or dried autoclaved poultry manure to diets containing crystalline essential amino acids

Female broiler chicks were offered semi‐purified diets containing crystalline essential amino acids, including proline, supplemented with various nitrogen sources. Individual final body weights, food consumption and food conversion efficiencies (FCE) were measured.

In the first experiment 70 1‐d‐old chicks were given one of seven diets for 21 d; basal (1A) or basal plus: 12% l‐glutamic acid (1B), 17.05% monoammonium citrate (1C), 9.23% diammonium citrate (1D), 6.61% triammonium citrate (TAC) (1E), 2.45% urea (1F) or 4.05% triammonium phosphate and 12.56% calcium lactate (1G). Using body weight at 21 d and FGE as measures of the effectiveness of the various nitrogen sources the orders were 1E (171 g), 1B (154 g) > 1D (131 g), 1G (123 g), 1F (118 g) >basal (89 g) >1C (66 g), (P < 0.05) and 1B (0.456), 1E (0.420) >1G (0.346), 1F (0.329), 1D (0.319) > basal (0.269) > 1C (0.192), (P < 0.05), respectively.

In the second experiment 70 7‐d‐old chicks were offered one of seven diets for 14 d; basal (2A) or basal plus 12% l‐glutamic acid (2B), 8.0% l‐glutamic acid and 2.21% TAC (2C), 4.0% L‐glutamic acid and 4.42% TAC (2D), 6.61% TAC (2E), 3.43% uric acid (2F) or 20.09% dried autoclaved poultry manure (2G). When the final body weights at 21 d and FCE were placed into a rank order, the degrees of effectiveness were: 2C (250 g), 2B (245 g), 2D (244 g), 2G (240 g) > 2E (206 g) > basal (165 g), 2F (150 g) (P < 0.045) and 2B (0.471), 2G (0.451), 2C (0.445), 2D (0.436) >2E (0.399) > basal (0.314) >2F (0.243) (P < 0.05), respectively. The results for diets 2B, 2C, 2D and 2E appear to indicate that 4% glutamic acid is required for maximal chick growth with these crystalline amino acid diets.     

牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

为获得牛支原体（Mycoplasma bovis,M.bovis）VspX蛋白单克隆抗体,将编码该蛋白的基因克隆、表达并纯化,作为免疫原,以QuickAntibody-Mouse 5W为免疫佐剂,免疫BALB/c小鼠。经3次免疫后,将小鼠脾细胞与SP2/0骨髓瘤细胞融合,经3次亚克隆筛选后,共获得5株能稳定分泌抗VspX蛋白抗体的杂交瘤细胞株,分别命名为1A8、3A3、3C12、3H9及4D11。亚型鉴定表明,3C12重链为IgG2b,其余4株为IgG1,轻链均为κ链。间接ELISA结果表明,5株细胞培养上清的抗体效价在1：1×10⁴~1：2×10⁵,腹水效价在1：1×10⁵~1：8×10⁵。选其中两株杂交瘤细胞株3H9和4D11的腹水纯化,进行亲和力测定,解离常数分别为6.3×10⁹和7.8×10⁹,属高亲和力抗体。Western blotting结果显示,5株单抗均能与牛支原体发生特异性反应,而单抗4D11与羊无乳支原体标准株PG2和丝状支原体丝状亚种标准株PG3均不反应。流式细胞术结果表明,单抗4D11与牛支原体表面的VspX的结合呈剂量依赖性。间接免疫荧光结果表明,单抗4D11可以识别黏附到胚胎牛肺细胞上的重组VspX蛋白。本试验成功制备的单克隆抗体为VspX蛋白功能的研究奠定基础。     

Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801

To obtain the monoclonal antibody (McAb) against VspX protein of Mycoplasma bovis (M. bovis),VspX gene was amplified, expressed and purified. Then, BALB/c mice were immunized subcutaneously three times with the purified recombinant VspX (rVspX) mixed with QuickAntibody-Mouse 5W adjuvant. Three days after the last injection, spleen cells were collected aseptically, and fused with SP2/0 myeloma cells in the presence of polyethylene glycol. By the clone selection, five stable hybridomas against VspX protein were obtained, separately named as 1A8, 3A3, 3C12, 3H9 and 4D11. Antibody titers in cell supernatant were from 1:1×10⁴ to 1:2×10⁵, while from 1:1×10⁵ to 1:8×10⁵ in ascites of mice by indirect ELISA. The subtypes were determined to be IgG1 and IgG2b class, and all light chains were κ chain. The affinity constant of McAb 3H9 and 4D11 were 6.3×10⁹ and 7.8×10⁹, respectively, and they belonged to high-affinity antibodies. Western blotting results showed that all of five McAbs could specifically react with M.bovis, however, McAb 4D11 could not react with Mycoplasma arginini PG2 and Mycoplasma mycoides subsp. PG3. Flow cytometry showed that McAb 4D11 reacted with surface VspX of M. bovis in a dose-dependent manner. Indirect immunofluorescence assay demonstrated that 4D11 McAb was able to detect rVspX protein binding to embryonic bovine lung cells. In the present study, McAbs against rVspX protein had been successfully prepared, which provided a basis for future researches about the function of VspX protein and the pathogenesis of M. bovis.     

Heterogeneity of human lymphocyte Fc receptors: Studies using heat-aggregated and antigen-complexed IgG from human, rabbit, guinea pig, horse and goat

We have studied the ability of human peripheral blood lymphocytes (HuPBL)⁴ to interact with IgG from several animal species. Three functions or activities that are reported to depend on an interaction between complexed IgG and HuPBL receptors (R) for the Fc piece of IgG (FcγR) were compared: (1) antibody-dependent cell-mediated cytotoxicity (ADCC); (2) binding of heat-aggregated IgG (aggG); and (3) rosette formation with IgG-sensitized erythrocytes [RBC-A(γ)]. IgG (and IgM) antibodies to chicken erythrocytes (CRBC) were purified from the sera of the following species after injection with CRBC stroma: (1) horse (Ho); (2) goat (Go); (3) rabbit (Ra); and (4) guinea pig (Gp). Good IgG-agglutinating antibody titers were obtained from each injected species.

Using ⁵¹Cr-labeled CRBC targets and HuPBL effector cells, only Ra anti-CRBC IgG gave good ADCC at high dilutions. Ho and Go anti-CRBC (IgG) failed to give A C , and p anti-CRBC (IgG) gave approx. 30% of the level of kill as Ra. Ra Fab'₂ fragments of IgG antibody failed to produce ADCC.

Treatment of HuPBL with Ra anti-lymphocyte serum (ALS) almost totally ablated ADCC, whereas HoALS failed to alter ADCC. Pretreatment of HuPBL with aggG showed that Ra or Hu aggG gave essentially equal inhibition of ADCC, Gp gave approx. 30% of the degree of inhibition as Hu and Ra, and Ho or Go aggG had essentially no effect of ADCC. These results confirmed the following order of ability of IgG to interact with HuPBL ADCC killer (K) cells: (Hu )Ra > Gp Ho, Go. The data suggest that Gp IgG interacts with only a subpopulation (≈ 30%) of HuPBL K cells.

The binding of aggG to total HuPBL failed to strictly correlate with the ADCC results or with the results of rosette formation between total HuPBL and CRBC-A(γ). The observations suggest that there is a heterogeneity of FcγR between K and non-K cell subpopulations of HuPBL both in terms of the type of complexed IgG they are able to bind, and in terms of the species of origin of the IgG. The data also support contentions that FcγR that bind RBCA(γ) complexes differ from those that bind aggG.     

Th1/Th2型免疫反应相关基因在巨噬细胞RAW 264.7应对细粒棘球绦虫原头蚴刺激时的表达谱研究

本研究旨在解析巨噬细胞RAW264.7在应对细粒棘球绦虫原头蚴刺激时其Th1、Th2型免疫反应相关基因的差异表达规律,为进一步揭示巨噬细胞抗细粒棘球绦虫原头蚴免疫调控机制奠定理论基础.将细粒棘球绦虫原头蚴和巨噬细胞RAW264.7共培养6、24、72 h,收集RAW264.7细胞,提取总RNA,构建cDNA文库,利用R...     

Influence of arachidonic acid metabolites and steroids on function of bovine polymorphonuclear neutrophils.

Polymorphonuclear neutrophils (PMN) from 4 ovariectomized healthy cows were incubated with 0 (control), 10(-8), 10(-7), and 10(-6) M arachidonic acid metabolites of the cyclo- and lipoxygenase pathways for 30 minutes, and with steroids for 2 hours. Immediately after incubation, PMN were subjected to the following function assays: chemotaxis against zymosan-activated serum, chemotaxis against arachidonic acid metabolite or steroid at the doses given (only control PMN were tested), random migration, ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus (125I-IdUR-S aureus), iodination of proteins, cytochrome C reduction, antibody-independent and -dependent cell-mediated cytotoxicity (AICC and ADCC). Prostaglandin F2 alpha was chemoattractant and stimulated ingestion of 125I-IdUR-S aureus. Prostaglandin E2 stimulated cytochrome C reduction, whereas prostacyclin inhibited iodination of proteins. Thromboxane B2 stimulated ADCC. Leukotriene B4 was chemoattractant for bovine PMN and stimulated random migration and AICC. 5-Hydroxyeicosatetraenoic acid was also chemoattractant, but inhibited ingestion of 125I-IdUR-S aureus. 15-Hydroxyeicosatetraenoic acid was chemoattractant and decreased ADCC. Lipoxin A4 stimulated random migration, whereas lipoxin B4 inhibited chemotaxis against zymosan-activated serum, but was chemoattractant and stimulated cytochrome C reduction. 12-Hydroxyhepadecatrienoic acid and 12-hydroxyeicosatetraenoic acid did not influence any of the PMN functions tested. Of the steroids tested, cortisol increased ADCC, and progesterone stimulated cytochrome C reduction, but decreased ADCC. 17 beta-Estradiol and estrone were chemoattractant and stimulated cytochrome C reduction. In addition, estrone also stimulated random migration.(ABSTRACT TRUNCATED AT 250 WORDS)