生长抑素和皮质抑素双表达DNA疫苗免疫杂交水牛犊牛的促生长效果研究

试验旨在探究生长抑素（SS）和皮质抑素（CST）双表达DNA疫苗免疫杂交水牛犊牛的促生长效果及疫苗本身的安全性。将鉴定正确的pVGS/2SS-2A-S/CST-asd质粒电转入减毒猪霍乱沙门氏菌C500构建SS和CST双表达活载体疫苗,喷鼻免疫杂交水牛犊牛后观察其对犊牛生长性能的影响。选取体重相近的2~6月龄杂交水牛犊公牛24头,随机分为低（TL）、中（TM）、高（TH）剂量组和PBS对照组（NC）,每组6头,初次免疫（简称初免）持续3 d,间隔2周后用相同剂量加免一次。结果显示,初免后2周便可产生抗SS和CST抗体,以TL组最佳,表现为更高的抗体水平和阳性率,7周时各试验组阳性率呈下降趋势;TL和TM组的平均日增重在初免后2和7周均高于对照组,但各组间差异均不显著（P>0.05）,而抗体阳性组（P）的平均日增重在0~2周显著高于阴性组（N）（P<0.05）。相关激素的检测结果显示,在初免后2和7周,试验组生长激素（GH）和胰岛素生长因子1（IGF-1）的水平均高于对照组,且以TL组表现最佳,除初免后7周GH与对照组无显著差异外,其余均极显著高于对照组（P<0.01）;初免后2周抗体阳性组极显著高于阴性组（P<0.05）。细胞因子检测结果显示,在初免后2周,试验组白细胞介素4（IL-4）含量高于对照组,且TL和TM组均极显著高于对照组（P<0.01）,尤以TL组最高,免疫后7周TL和TH组均显著高于对照组（P<0.05）,而免疫后2周对照组免疫干扰因子γ（INF-γ）含量极显著高于试验组（P<0.01）,免疫后7周对照组INF-γ含量显著低于TL和TH组（P<0.05）;阳性组IL-4水平在免疫后2周、INF-γ水平在免疫后7周均显著高于阴性组（P<0.05）,其他各组间差异不显著（P>0.05）。对血清中血糖（GLU）、总蛋白（TP）、总胆固醇（CH）和尿素氮（BUN）水平进行测定发现,除免疫后2周总蛋白含量TL组显著高于对照组外（P<0.05）,其他指标各组间差异均不显著（P>0.05）。对免疫后的水样、土样和粪样进行目的基因扩增发现,在PCR灵敏度范围内均未检测到,证实了疫苗的安全性。由上述结果可知,疫苗免疫后能取得良好的免疫效果,使机体产生显著的免疫应答,以低剂量组效果最佳,且没有对环境产生不良影响。     

以减毒沙门菌为载体的SS和CST双表达DNA疫苗经不同途径免疫犊牛的效果评价

旨在探讨SS和CST双表达DNA疫苗(以下简称双表达DNA疫苗)经不同途径免疫犊牛的效果。本研究选择27头2~6月龄的奶水牛犊牛,随机分成4组,前3组分别采用口服(O组)、喷鼻(N组)、口服+喷鼻(ON组)的方式接种双表达疫苗,剂量均为3×10⁷ CFU·mL^-1,对照组(C)使用PBS进行口服+喷鼻免疫,并于2周后加强免疫一次。结果表明,3种免疫方式均可诱导犊牛产生SS抗体和CST抗体;初免后2周,N和ON组阳性率最高,均为85.71%;6周时,各组阳性率均下降,O和N组的抗体水平也逐渐下降,但ON组的抗体水平升高;10周时,ON组阳性率下降到14.29%,O和N组均检测不到抗体。从增重来看,2周时,ON组的平均日增重显著高于O、N和C组(P＜0.05),在整个试验周期(0~10周),与C组相比,O、N和ON组的增重分别提高了5.26%、10.53%、15.79%;初免后10周,ON组的胸围、腿臀围均显著高于C组(P＜0.05)。血清中生长相关激素检测结果表明,各组GH、IGF-1、T₃的含量,ON＞N＞O＞C组;其中,2周时,O、N和ON组IGF-1的浓度显著高于C组(P＜0.05);6周时,ON组T₃和T₄的浓度均显著高于O和C组(P＜0.05)。细胞因子检测结果表明,3种免疫方式都能激发机体产生体液免疫和细胞免疫应答,6周时,ON组INF-γ的含量显著高于C组(P＜0.05),其余各组之间差异不显著(P＞0.05)。血生化指标检测结果表明,各组之间,犊牛血清中总蛋白、葡萄糖、游离脂肪酸和尿素氮的含量差异不显著(P＞0.05)。综上所述,口服、喷鼻和口服+喷鼻3种方式免疫犊牛均可产生良好的效果,尤其以口服+喷鼻联合免疫效果最佳。     

牛分枝杆菌三价组合及融合DNA疫苗免疫效果的研究

分泌性蛋白Ag85B、MPB64和ESAT-6为Mycobacterium boris的主要保护性抗原,在诱导机体免疫反应和抵抗感染中发挥重要作用。本研究以peDNA3．1（＋）为载体,构建了由上述3种抗原基因构成的不同疫苗：3基因融合（peDNA-MPB64-Ag85B-ESAT-6,pCMAE）DNA疫苗和三价（pcDNA-Ag85B＋peDNA-MPB64＋pcDNA-ESAT-6）DNA疫苗,评价各种DNA疫苗诱导的体液免疫、细胞免疫及以BCG攻毒后的免疫保护水平。试验结果表明,多基因融合DNA疫苗免疫后的小鼠血清抗体水平、淋巴细胞增殖（SI值）、gamma interferon（IFN-γ）和interleukin-2（IL-2）水平明显高于其他各组（P〈0．05）,攻毒保护效果也优于多价DNA疫苗,达到了BCG疫苗的免疫保护水平,表明本研究制备的融合DNA疫苗具有良好的应用前景。     

牛分枝杆菌二价组合及融合DNA疫苗的免疫效果

利用PCR技术和重叠延伸剪接(SOE)技术扩增牛分枝杆菌ag85b、mpb64基因和ag85b-mpb64融合基因,连接真核表达载体pCDNA3.1(+),构建二基因融合(pCDNA-MPB64/Ag85B,pCMA)和二价组合(pCDNA-Ag85B+pCDNA-MPB64,pCA+pCM)DNA疫苗.以牛分枝杆菌卡介苗(BCG)为阳性对照,以pCDNA3.1(+)和PBS为阴性对照,免疫BALB/c小鼠,检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN-γ及IL-2分泌情况.结果显示:融合DNA疫苗组免疫小鼠血清抗体水平、刺激值(SI值)及IFN-γ和IL-2的分泌水平均明显高于其他各组(P<0.05).二价组合DNA疫苗组的体液和细胞免疫指标与BCG组相当(P>0.05),而显著高于两阴性对照组(P<0.05).     

肌肉生长抑素基因工程疫苗免疫动物的效果研究

为检验肌肉生长抑素基因工程疫苗对动物生长的影响,用由大肠杆菌表达的肌肉生长抑素蛋白与油佐制成疫苗,肌肉注射免疫25天龄小白鼠、并观察生长状况,每周称重,纪录结果,饲养至95天龄后试验结束;肌肉注射免疫21天龄幼鸡,饲养至98天龄后进行屠宰,分离胸肌、腿肌并称重。结果,与对照组相比,注射肌肉生长抑素基因工程疫苗的小白鼠体重最高增加17.17%;鸡体重最高增加14.27%,免疫效果比较理想,达到了预期的目的。     

河流型水牛杂交改良德昌水牛的效果初报

为观察河流型水牛(摩拉水牛和尼里-拉菲水牛)与德昌水牛的杂交改良效果,笔者对杂交一代牛初生、1周岁、2周岁的体尺、体重进行测定,分析生长发育情况;同时测定杂交一代母水牛的产奶量、泌乳期,分析产奶性能情况.结果显示:杂交一代牛的平均初生重为42.58±6.32 kg,1周岁体重为285.22±15.09 kg,2周岁体重...     

牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因，连接真核表达载体pcDNA3．1（＋），构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠：pCE6组、pC70—83-E6组、pcDNA3．1（＋）和PBS对照组，采用间接EI。ISA法检测免疫小鼠血清特异性抗体水平，MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN—y分泌情况。结果表明，2重组质粒免疫组小鼠的血清抗体水平持续上升，而2对照组始终维持在较低水平，且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后，pCE6组和pC70—83-E6组小鼠的SI值与2对照组均差异显著（P〈0．05），2重组质粒免疫组间差异不显著（P〉0．05）；2重组质粒免疫小鼠脾细胞产生的IFN—y均显著高于2对照组（P〈0．05），且pC70-83-E6组明显高于其他3组（P〈0．05）。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。     

生长抑素基因工程疫苗对大鼠生长及免疫的影响

以生长抑素基因工程疫苗免疫离乳大鼠和 12 0 g左右大鼠 ,结果该疫苗能明显促进 12 0 g左右大鼠的生长 ,但对离乳大鼠没有明显效果。 2组处理均发现 ,大鼠血清生长激素水平显著升高 ,但是前者血清 IGF- 1水平没有出现明显变化。结果提示 ,生长抑素基因工程疫苗的促生长作用与动物的生长周期密切相关 ,而且这种状况很可能由 IGF- 1水平所决定。该疫苗免疫大鼠引发诸多免疫指标的改变 ,推测生长抑素基因工程疫苗通过调节激素变化间接地影响了动物的免疫功能。     

沼泽型水牛卵泡抑素基因的真核表达研究

试验通过候选基因法,选定卵泡抑素(follistatin,FS)作为影响水牛繁殖性能的主要候选基因,通过基因工程的方法,用XhoⅠ、SacⅡ双酶切广西大学动物遗传育种与繁殖实验室克隆的添加有ACC的Kozaka 序列的pMD-bFS质粒和pEGFP-N₁,构建pEGFP-bFS真核表达质粒,将其与阳离子脂质体混匀后,分别转染体外培养的水牛胎儿成纤维(BFF)细胞和仓鼠肾(BHK)细胞系,经过48 h培养后,在相差荧光显微镜下观察绿色荧光蛋白的表达水平,用RT-PCR和Western blotting方法对转入质粒表达进行定性鉴定。结果显示,本研究成功构建了添加有ACC的Kozaka 序列的pEGFP-bFS真核表达质粒,该重组质粒在BFF和HBK两种细胞均表达,但在HBK细胞系的表达量稍高。本研究结果将为下一步制备具有生物活性的bFS工程疫苗,研究bFS对水牛繁殖性能的影响提供参考。     

口蹄疫病毒重组鸡痘病毒活载体疫苗和DNA疫苗免疫牛的试验研究

将本课题组构建的共表达FMDV衣壳蛋白前体P1—2A基因以及蛋白酶3C基因的重组鸡痘病毒活载体疫苗vUTAL3CP1以及共表达P1—2A和猪白介索18基因的重组DNA疫苗（pVIRIL18P1），分别以单独及混合的共3种方式接种牛，然后通过间接ELISA、中和试验和T淋巴细胞增殖试验评价其诱导的特异性体液和细胞免疫水平。结果表明这2种基因工程疫苗均能诱导牛产生特异性的体液及细胞免疫应答。其中重组鸡痘病毒vUTAL3CP1免疫组以及联合免疫组（vUTAL3CP1／pVIRIL18P1，pVIRIL18P1／vUTAL3CP1）诱导的中和抗体滴度分别达到1：64、1：64和1：54，已接近于常规灭活疫苗水平（1：90），而特异性的T淋巴细胞增殖反应则比后者高得多（P〈0．01）。该研究结果为进一步进行免疫攻毒试验，并最终筛选出最佳疫苗和免疫程序奠定了基础。     

Study on the Growth Promoting Effect of Crossbred Buffalo Calves Immunized with DNA Vaccine of Co-expression of Somatostatin and Cortistatin

The purpose of this study was to investigate the growth promoting effect and the safety of the vaccine in crossbred buffalo calves immunized with somatostatin and corticostatin co-expression DNA vaccine.The identified plasmid of pVGS/2SS-2A-S/CST-asd was electroporated into attenuated Salmonella Choleraesuis C500 to construct the co-expression live vector vaccine of somatostatin and corticostatin,and evaluate the effect on the growth performance of the crossbreds buffalo after nasal immunization.Twenty four 2-6 months of age crossbred buffalo calves with similar body weight were randomly divided into 4 groups immunized with the vaccine in low dosage group (TL),middle dosage group (TM),high dosage group (TH) and negative control group (NC).Immunization was performed once a day for 3 consecutive days,and a booster was given 2 weeks late.The results showed that the antibody of SS and CST could be produced on 2 weeks after the primary immunization,and TL group was the best,showing higher antibody level and positive rate.At 7 weeks,the positive rate of experimental group showed a downward trend.The daily gain of each experimental group was higher than that of control group at 2 and 7 weeks after the primary immunization,but there was no significant difference between experimental groups (P>0.05),while the average daily gain of antibody positive group (P) was significantly higher than that of negative group (N) at 2 weeks (P<0.05).The results of related hormones showed that the levels of growth hormone (GH) and insulin growth factor 1 (IGF-1) were higher in experimental groups at 2 and 7 weeks after the primary immunization.In each experimental group,TL group showed the best performance,both extremely significantly higher than that of control group except GH level at 7 weeks after the primary immunization (P<0.01),antibody positive group was also significantly higher than negative group (P<0.05).Cytokine test results showed that the interleukin 4 (IL-4) level in experimental groups at 2 weeks after the primary immunization was higher than that of control group,and the IL-4 level in TL and TM groups was extremely significantly higher than that of control group (P<0.01),especially in TL group.The IL-4 level in TL and TH groups at 7 weeks after the primary immunization was significantly higher than that of control group (P<0.05).At 2 weeks after the primary immunization,the level of immune interferon γ (INF-γ) in control group was extremely significantly higher than that of experimental groups (P<0.01),but at 7 weeks after immunization was significantly lower than that of TL and TH groups (P<0.05).The levels of IL-4 and INF-γ in antibody positive group were significantly higher than that of negative group at 2 and 7 weeks after the primary immunization (P<0.05),respectively,and there was no significant difference among other groups (P>0.05).The levels of blood glucose (GLU),total protein (TP),total cholesterol (CH) and urea nitrogen (BUN) in serum showed that except that the level of total protein in TL group at 2 weeks after the primary immunization was significantly higher than that of control group (P<0.05),there was no significant difference of other indexes among each group (P>0.05).In order to confirm the safety of the vaccine,the genes of water,soil and fecal samples after immunization were amplified,which were not detected in PCR sensitivity range.The results indicated that the vaccine could achieve a good immune effect,make the body produce a significant immune response,and the effect of the low dosage group was the best.The vaccine did not cause adverse effects on the environment.     

西杂牛产奶性能试验研究

用西门塔尔级进杂交改良本地黄牛,产生西杂一代、二代、三代牛。分别对其1、2、3胎产奶量进行测定,结果表明：各代胎次之间奶量差异不显著（P〉0.05）,但从1胎到3胎上升势头明显,3胎最高;各代305 d平均产奶量差异极显著（P〈0.01）,西杂一代最低、三代最高,产奶天数随着杂交代次提高而逐渐增加;西杂牛产奶收益显著。开展西杂牛挤奶是增加奶源、提高养殖效益的重要举措。     

犬细小病毒核酸疫苗、重组活载体疫苗与弱毒疫苗免疫犬实验研究

分别用犬细小病毒（CPV）核酸疫苗（pVCPV-VP2）、CPV重组活载体疫苗（CAV2／CPV）与CPV弱毒疫苗对犬进行了免疫试验,以检测不同CPV疫苗的免疫原性。采用CPVELISA、CPVHI与CPV微量中和试验检测免疫犬的体液免疫水平,采用淋巴细胞转化试验检测犬的细胞免疫水平。结果,pVCPV—VP2和CAV2／CPV均能诱导机体产生抗CPVELISA抗体与抗CPV中和抗体,但是pVCPV-VP2不能诱导机体产生可检测的抗CPVHI抗体,而CAV2／CPV能够诱导机体产生抗CPVHI抗体。淋巴细胞转化试验结果,pVCPV-VP2和CAV2／CPV免疫犬的外周血淋巴细胞对ConA与CPV的刺激均出现明显的增殖反应。结果表明,pVCPV—VP2和CAV2／CPV免疫犬均能诱导机体产生抗CPV的特异性体液免疫反应和细胞免疫反应,两者所表达的VP2蛋白均具有较好的免疫原性。CAV2／CPV以及pVCPV—VP2和CAV2／CPV联合免疫犬的抗CPV体液免疫水平和细胞免疫水平均比用pVCPV—VP2单独免疫犬的体液免疫水平和细胞免疫水平高。但CAV2／CPV诱导机体产生的抗CPV特异性免疫反应仍然比CPV弱毒疫苗诱导机体产生的抗CPV特异性免疫反应弱。另外,CAV2／CPV还能诱导机体产生抗CAV-2的特异性免疫反应。     

苜蓿草颗粒育肥西杂牛效果试验

在基础日粮相同的情况下,试验组采用苜蓿草颗粒,对照组采用铡碎的苜蓿干草,对12头1岁西杂公牛进行60 d育肥试验.测定分析了两组牛的体重、日增重及经济效益,以比较苜蓿的不同调制方法对育肥效果的影响,试验结果表明:头均日增重试验组0.95 kg,比对照0.72kg提高了31.84%(P＜0.01),经济效益试验组比对照组提高了54.3%(P＜0.01).因此选择苜蓿草颗粒进行西杂公牛的育肥效果较好.     

营养水平和育肥期对西杂牛肥育的影响

该试验选择20～24月龄左右（体重400±10kg）的西杂牛（西门塔尔6×本地黄牛早）40头。试验设计4个组,每组为10头,育肥期分别为90d和120d,以青贮玉米和酒槽为基础粗料,经过3个月的短期集中肥育,研究杂种肉牛肥育效果与不同肥育期和营养水平的关系,进而分析各项屠宰指标。结果表明：饲喂1料组的平均日增重和饲料转化率均优于饲喂Ⅱ料组,且肥育90d即可达到理想育肥效果。     

杂交组合对肉牛育肥效果的影响

试验旨在研究杂交组合对肉牛育肥效果的影响。采用单因素分组试验设计,选取体重和年龄相近、生长发育正常的西门塔尔牛×科尔沁牛（西科,A组）、夏洛莱牛×科尔沁牛（夏科,B组）及科尔沁牛（C组）各10头公牛,正试期95 d后,测定其体重,屠宰、肉品质等指标,并核算经济效益。结果显示,杂交对采食量无显著影响（P>0.05）,但杂交组牛料重比、肌肉脂肪含量显著低于科尔沁牛（P<0.05）,杂交组牛的胴体重、净肉重、胴体率及前腿、腹肉、脊肉和后腿重量均显著高于科尔沁牛（P<0.05）,各组之间16种氨基酸含量差异不显著（P>0.05）,西科、夏科杂交牛及科尔沁牛之间棕榈油酸、硬脂酸、亚油酸含量存在差异。结果表明,在本试验条件下,杂交组牛育肥效果和经济效益好于科尔沁牛,但科尔沁牛的脂肪和不饱和脂肪酸含量高于杂交组合牛;杂交组合对肉牛的综合育肥效果更好。     

Effect of Different Crossbred Combinations on Fatting Performance in Beef Cattle

This study was aimed to determine the effects of different crossbred combinations on fatting performance in beef cattle. Using single factor randomized block design, 30 bulls (health, similar weight and age) were chosen, Simmental cattle×Kerqin beef cattle (group A), Charolais cattle×Kerqin crossbred beef cattle (group B) and Kerqin beef cattle (group C) were divided into three groups with 10 bulls per group. After 95 d, the body weight, slaughter performance, meat quality and the economic benefits of fattening were measured. The results showed that there were no significant influence on dry matter intake among trials (P>0.05). The F/G, ethanolic extract (EE) of muscle in crossbred groups (groups A and B) were significant lower than group C (P<0.05). The weight of carcass, weight of meat, rate of carcass, and the weight of shank, plate, chine and rump in crossbred groups were significant higher than group C (P<0.05). There were no significant difference in content of amino acids among groups (P>0.05),and it had difference in the content of palmitoleic acid (PAA),stearic acid(SA) and linoleic acid (LA). In this trial, it had better fattening performance and the economic benefits in crossbred cattle groups than Kerqin beef group cattle, but the ethanolic extract and unsaturated fatty acids of muscle in Kerqin beef cattle were higher than the crossbred groups, and it had better fattening performance by crossbred.