抑制素基因免疫京白鸡后在心脏组织中的表达

[目的]探讨基因免疫的安全性。[方法]抑制素基因免疫京白鸡后,取其心脏组织制备石蜡切片,再利用免疫组化方法检测心脏组织切片中是否有抑制素基因残留（即是否有阳性点的存在）。[结果]在心脏组织切片中,试验组和对照组均无阳性点。[结论]抑制素免疫后京白鸡的心脏组织中无外源抑制素表达,说明抑制素基因免疫是安全的。     

细菌DNA和gm-csf基因对抑制素基因免疫的作用

40只小鼠分为4组,分别注射pC ISI(抑制素质粒)、pC ISI+细菌DNA、pC ISI+pGM-CSF和生理盐水(8.5 g.L-1)。pC ISI剂量为20μg,与佐剂等量混合,2周后加强免疫1次。试验结果表明,细菌DNA佐剂组诱导77.8%(7/9)的个体产生了抑制素阳性抗体,抗体以IgG2α型为主,gm-csf佐剂组产生的抗体以IgG1型为主;基因佐剂联合抑制素基因免疫激发的淋巴细胞增殖能力显著高于抑制素基因单独免疫组(P<0.05);细菌DNA佐剂组阳性鼠的动情期血浆雌二醇高于阴性鼠(P<0.05),免疫对动情期孕酮水平无显著影响(P>0.05)。上述结果表明,细菌DNA和gm-csf能增强抑制素基因的免疫效果。     

鸡瘦蛋白基因克隆中的若干问题研究

根据GenBank中鸡的LeptinmRNA序列(AccessionNo.AF012727)设计12对引物,用RT-PCR方法从不同品种(系)、不同时期及不同处理鸡的脂肪、肝脏和卵巢组织总RNA中没有扩增出鸡的Leptin基因片段;根据鸡的EST数据库中查到的一条鸡Leptin基因前体序列设计4对引物,用RT-PCR方法在包括卵巢在内的多个组织的cDNA中没能扩增出正确序列。为提高鸡Leptin基因的表达水平,通过给鸡注射胰岛素,利用RT-PCR方法对肝脏、卵巢和脂肪组织的总RNA进行扩增,没有得到目的序列。根据已发表的哺乳动物的Leptin基因序列设计兼并引物对鸡基因组DNA和脂肪、肝脏组织的cDNA进行PCR,结果没有特异性扩增条带,但在小鼠的基因组中可以获得稳定的扩增条带。用扩增长片段LATaq酶从鸡基因组DNA中也没有扩增出Leptin基因片段,而从小鼠的基因组DNA中,可扩增出小鼠Leptin基因片段;以小鼠LeptincDNA片段为探针,对鸡脂肪组织和肝脏组织来源的总RNA进行NorthernBlot分析,并未获得杂交信号;以猪的LeptincDNA片段为探针,对鸡基因组DNA进行SouthernBlot分析,并未获得特异性的结果。研究结果表明,在鸡的脂肪、肝脏和卵巢组织中不存在与小鼠Leptin基因同源性如此高的mRNA序列,在鸡的基因组中也不存在与小鼠、猪等哺乳动物Leptin基因序列同源性如此高的基因?     

转基因大豆对雄性鼠生殖系统的安全性评估

以含转基因豆粕饲料喂食小鼠,观察对雄性小鼠生殖系统的影响。喂食小鼠120d后,通过PCR方法检测外源基因在睾丸中的存在情况;对睾丸组织制作石蜡切片,观察睾丸组织的病理损伤情况;通过流式细胞术检测睾丸组织中各生殖周期细胞比例;通过彗星电泳检测小鼠精子DNA损伤情况。结果表明:在睾丸组织中未检测到外源基因;睾丸组织切片未发现明显的病理学变化;试验组与对照组小鼠睾丸组织中各生殖周期精细胞比例无显著差异;未检测到精子DNA损伤。说明喂食转基因饲料对小鼠的生殖系统无显著影响。     

抑制素α(1～32)基因免疫对大鼠卵泡发育和生殖激素的影响

 选择 40只 3 0日龄雌性大鼠分为 4组 ,分别用含重组抑制素α(1～ 3 2 )基因真核表达质粒pcINH 0 μg(对照组 )、15μg(试验组 1)、2 5μg(试验组 2 )、40 μg(试验组 3 )的脂质体进行主动免疫 ,间隔 2 0d后用不含脂质体的重组基因质粒加强免疫 1次。结果发现 ,抗体阳性鼠占 50 % (13 / 2 6) ,但加强免疫 10d后的抗体P/N值没有升高 ,而且免疫剂量的增加并没有提高阳性鼠的比例 ;抗体阳性鼠卵巢上的成熟卵泡数比阴性鼠多 2 .3 ,但统计差异不显著 (P >0 .0 5)。首次免疫 10d后阳性鼠血浆FSH浓度显著升高 (P <0 .0 5) ;但血浆雌二醇水平无显著差异 (P >0 .0 5)。加强免疫 10d后血中促卵泡素水平没有升高。本试验结果证明 ,应用重组抑制素真核表达质粒免疫动物可以产生抗抑制素抗体。     

异种器官移植转基因猪检测系统的建立

以 PCR、免疫荧光、细胞毒试验在 DNA、表达、功能水平对转 h DAF基因猪进行了检测。结果表明 :转基因猪的整合率为 19.35 % ( 18/ 93) ;在 14头转基因阳性猪中 ,有 7头动脉肌肉层表达了外源 h DAF基因 ,6头猪的外源基因表达产物可以抑制猪淋巴细胞的裂解     

鸡YAP1基因在不同生长发育时期卵泡中的时空表达分析

为探讨YAP1基因在鸡卵泡生长发育中的调控作用,以150 d海兰褐蛋鸡为供试材料,采用相对定量RT-PCR方法对YAP1基因在鸡卵巢组织不同发育时期卵泡中的mRNA转录水平进行检测分析,并用免疫组织化学方法对YAP1蛋白质分子在鸡前等级卵泡中的时空表达模式进行蛋白定位分析。结果表明:鸡YAP1基因mRNA在卵巢间质、前等级卵泡和不同等级化的卵泡中均有表达,但在不同等级的卵泡中表达差异不显著(P0.05),呈现出组成型表达的特点。鸡YAP1蛋白质分子主要在卵巢间质组织、前等级卵泡卵母细胞和颗粒细胞中有表达,但在膜层细胞中未检测到其表达信号。此结果表明,YAP1分子可能主要以自分泌或旁分泌的方式在前等级卵泡的生长发育过程中发挥着重要调节作用。     

异种器官移植转基因猪检测系统的建立

以PCR、免疫荧光、细胞毒试验在DNA、表达、功能水平对转hDAF基因猪进行了检测。结果表明：转基因猪的整合率为19．35％（18／93）；在14头转基因阳性猪中，有7头动脉肌肉层表达了外源hDAF基因，6头猪的外源基因表达产物可以抑制猪淋巴细胞的裂解。     

抑制素免疫对崇明白山羊公羔羊生殖发育的影响

利用重组抑制素对崇明白山羊雄性断奶羔羊(n=6)在第0天、第21天、第42天、第63天共进行4次免疫,探讨抑制素免疫对其睾丸发育、激素水平及生殖相关基因表达量的影响。结果表明:试验期间,免疫组抑制素抗体水平均显著高于对照组;在试验的第84天阉割时,免疫组和对照组试验羊在体重[(21.87±2.19)kg vs(23.58±5.13)kg]、阴囊周长[(20.95±0.74) cm vs (21.57±1.65) cm]、睾丸质量[(56.88±4.96) g vs(62.99±4.17)g]方面无显著差异;睾丸组织石蜡切片分析表明,免疫组和对照组试验羊在睾丸组织学上没有显著差异;血清卵泡刺激素(FSH)及睾酮(T)水平在二免后7 d免疫组显著高于对照组,其余采样时间无显著差异;血清促黄体生成素(LH)水平免疫组和对照组无显著差异;qRT-PCR检测结果表明,免疫组睾丸组织染色体联会相关基因Dmrtc2表达量极显著高于对照组(3.04 vs 1.00),性腺发育相关基因SOX30基因表达量显著高于对照组(1.48 vs 1.00),雄激素受体基因AR表达量显著低于对照组(0.59 vs 1.00),而FSH受体基因FSHR、LH受体基因LHR、精子发生相关基因GPx4以及减数分裂相关基因DDX4基因表达量两组之间没有显著差异。综上所述,抑制素免疫能短暂提高崇明白山羊FSH及T水平,提高睾丸Dmrtc2、SOX30基因的相对表达量,但对体重、LH激素水平、睾丸质量、睾丸组织学形态、睾丸FSHR、LHR等基因水平无显著影响。     

FoxO3转录因子在鸡卵巢组织中的表达鉴定

[目的]明确FoxO3转录因子在不同日龄鸡卵巢组织中的表达模式及其具体定位情况,为后续开展家禽卵泡激活及发育调控等相关机理研究提供科学依据.[方法]在GenBank中搜索鸡与其他物种(鸭、鹅、老鼠、猪、牛及人类)的FoxO3氨基酸序列,通过LaserGene分析鸡FoxO3氨基酸序列与其他物种FoxO3氨基酸序列间的亲缘关系及同源差异情况;利用RT-PCR鉴定FoxO3基因是否在鸡卵巢组织中表达,再采用实时荧光定量PCR检测不同发育阶段鸡卵巢组织中FoxO3基因的表达水平,最后以免疫荧光检测FoxO3蛋白在鸡卵巢组织中的定位情况.[结果]鸡与鸭和鹅的FoxO3氨基酸序列相似性分别为92.7%和95.3%,基于FoxO3氨基酸序列相似性构建的系统发育进化树也显示鸡与鸭和鹅的亲缘关系较近,说明FoxO3基因的进化相对较保守.在不同发育阶段的鸡卵巢组织中均能检测到FoxO3基因表达,且FoxO3基因在0日龄鸡卵巢组织中的相对表达量最高,显著高于在其他发育阶段的相对表达量(P<0.05).在0日龄鸡卵巢组织中未检测到FoxO3蛋白,但在21日龄和成年鸡的卵巢组织中均能检测到FoxO3蛋白;在21日龄鸡卵巢组织中FoxO3蛋白主要定位在原始卵泡细胞周围,在卵泡内部没有表达;而在成年鸡卵巢组织中FoxO3蛋白仅定位于大卵泡细胞边缘,小卵泡细胞内并未发现FoxO3蛋白.[结论]由于鸡和哺乳动物的FoxO3氨基酸序列高度同源,且FoxO3蛋白在鸡卵巢组织中的定位与在哺乳动物卵巢组织中的定位相似,说明FoxO3在鸡卵巢组织中发挥着与哺乳动物相似的功能,即在卵泡激活与成熟过程中发挥重要作用,因此通过调控FoxO3能有效提高鸡的繁殖性能.     

非抗性筛选抑制素基因疫苗免疫对奶牛产后子宫复旧的影响

将24头产后7 d的荷斯坦奶牛随机分为4组,高、中、低剂量免疫组(记为T1、T2、T3)分别注射1010、109、108 CFU/mL无抗性基因的抑制素真核表达质粒基因疫苗(重组菌C500(pXAIS))各3 mL,对照组(CK)注射10%生理盐水3 mL。初次免疫28 d后进行加强免疫。用B超诊断仪检测产后奶牛子宫恢复情况。结果表明:加强免疫能提高抗抑制素抗体P/N值,各免疫组P/N值均高于对照组,其中T1和T2组均与对照组差异显著(P0.05),加强免疫7 d,后T1组抗体阳性率最高,达83.33%;抑制素基因疫苗免疫30 d内,前21 d子宫颈和子宫孕角恢复较快,后7 d恢复较慢,各免疫组奶牛产后子宫颈恢复时间短于对照组,但差异不显著(P0.05);各免疫组产后子宫孕角恢复时间比对照组短,且T1组的恢复时间(28.75±1.92)d)与对照组((33.75±1.09)d)差异显著(P0.05)。以上结果表明,在本试验条件下,非抗性筛选的抑制素真核表达质粒基因疫苗免疫产后奶牛,加强免疫后能引起较好的免疫应答,且能促进奶牛产后子宫复旧。     

抑制素基因免疫黄牛的黄体发育与免疫纯度及剂量的关系

为探讨双拷贝抑制素pcISI基因疫苗纯度和剂量对肉牛黄体发育的影响,将150头同期发情的南阳黄牛随机分为8个试验组和2个对照组,试验组用pcISI按2种纯度、4种剂量(0.75、1.50、2.25、3.00 mg/头)进行免疫处理,2个对照组分别用空质粒和生理盐水进行处理;初次免疫21 d后进行加强免疫。结果表明:无论用高纯度还是低纯度疫苗免疫,2.25 mg/头剂量处理对黄体的作用效果最明显,且抗体水平与黄体发育密切相关,2.25 mg/头抑制素融合表达质粒组的黄体显著大于0.75 mg/头组(P<0.05),抗体阳性牛两侧黄体显著比抗体阴性牛的大(P<0.05)。     

双拷贝抑制素基因免疫对肉牛卵泡和黄体的影响

 【目的】探讨不同剂量的双拷贝抑制素pcISI基因免疫对肉牛生殖能力的影响。【方法】将58头同期发情肉牛随机分为6组, 每组肉牛分别注射0.75（T1）、1.5（T2）、2.25（T3）、3.0 mg/头（T4）pcISI质粒、3.0 mg/头 pcMV-S空质粒（C1）和3 mL/头 生理盐水（Ｃ2）,初次免疫21d后进行加强免疫,以探讨抑制素pcISI基因疫苗对肉牛卵泡和黄体发育的影响。【结果】4个剂量组的大卵泡数均高于对照组,除了T1外,其它3个剂量组均与对照组差异显著（P＜0.05）;T3和T4的中卵泡数均高于对照组,且差异显著（P＜0.05）;T3和T4的小卵泡数均高于对照组,且差异显著（P＜0.05）。无论左侧还是右侧,各剂量组成熟卵泡直径均高于对照组,除了T1外,其它3组两侧成熟卵泡均与对照组差异显著（P＜0.05）。无论左侧还是右侧,4个剂量组黄体直径均高于对照组,且均与对照组差异显著（P＜0.05）;T3黄体最大,T1最小,且T3与T1差异显著（P＜0.05）。加强免疫后10 d（BM10）抑制素抗体与成熟卵泡大小相关显著（r＝0.629,P＜0.05）,与黄体大小相关极显著（r＝0.651,P＜0.01）。【结论】双拷贝抑制素pcISI基因免疫可促进肉牛卵泡和黄体发育,2.25mg为最佳免疫剂量,抑制素抗体水平与卵泡和黄体发育有一定的相关性。     

Localization of Inhibin in Some Rat Tissues and Exploration of Its Transport Mechanism

The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism of inhibin by investigating the localization of inhibin and α subunits in the central nervous system （mainly in hypothalamus and pituitary） of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its α subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin α subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and α subunit could be transported through the BBB by the method of “separation and reconstruction”.     

主动免疫抑制素提高奶牛超数排卵胚胎产量的研究

 【目的】研究免疫抑制素提高奶牛超数排卵的效率以提高胚胎产量。【方法】免疫组首次免疫抑制素α亚基融合蛋白（1 mg?头-1）与白油佐剂混合乳液,首免后第28、56 d分别进行2次加强免疫,剂量为0.5 mg?头-1。对照组同时注射生理盐水与白油佐剂的混合乳液。在第二次加强免疫后10 d,采用FSH四日递减法进行超排处理,并根据母牛发情表现进行人工授精,试验一使用常规冷冻精液输精,试验二使用分离的雌性精子输精,输精后第7天进行冲胚。【结果】主动免疫抑制素能够有效提高血液抗抑制素抗体效价。试验一,免疫抑制素组头均获胚/卵总数为（15.8±2.8）枚,显著高于对照组（8.3±1.5）枚（P＜0.05）;头均可移植胚胎数为（9.6±3.1）枚,稍高于对照组的（5.8±1.6）枚（P＞0.05）。试验二,免疫抑制素组发情时间早于对照组(P＜0.05);输精后第7天血液孕酮浓度显著高于对照组（P＜0.05）;头均获胚胎总数和头均可移植胚胎数分别为（15.4±1.9）枚和（5.7±0.7）枚,显著高于对照组的（9.1±1.2）枚和（3.1±0.5）枚(P＜0.05);免疫组获得的一级胚胎为（3.8±1.0）枚,占总可移植胚数的58%,显著高于对照组的（0.6±0.2）枚或19.8%(P＜0.05)。【结论】主动免疫抑制素能够促进牛卵泡发育、提高超排效率和胚胎产量,在使用性控精液输精时不仅大幅提高雌性胚胎数量,同时大幅提高胚胎质量。     

Endocrine and ovarian responses in water buffalo cows immunized against inhibin and subjected to the Ovsynch protocol

The aim of this study was to investigate the feasibility of stimulating ovarian follicle development in order to improve fertility in water buffalo cows by immunization against inhibin. The experiment was carried out in early summer(May) and included 24 multi-parity crossbred Murrah-Swamp buffaloes that were divided into immunized(n=11) and control(n=13) groups. Each immunized cow was administered with a 2-m L immunogen of mineral oil adjuvant containing 2 mg of recombinant inhibin α-subunit fusion protein. The controls were treated with the adjuvant only. All animals received Ovsynch protocol treatment, starting on the day of the antigen administration, and they were artificially inseminated upon behavioral estrus. As a result, all of the immunized buffaloes generated antibodies against inhibin during the experimental period and had higher plasma concentrations of follicle-stimulating hormone(FSH), activin, and estradiol(E2) related to estrous expression. A higher proportion of immunized animals expressed estrus behavior than did the controls(72% vs. 30%, P0.05). On average, inhibin-immunized buffaloes had significantly more large follicles(≥9 mm in diameter) than the controls(mean±SEM; 1.2±0.1 vs. 0.84±0.1, respectively; P0.05) and a slightly higher mean total number of follicles(≥2 mm; 11.4±0.7 vs. 9.0±1.1, respectively; P=0.09) and small(2–4 mm) follicles(8.81±0.6 vs. 6.84±1.0, respectively; P=0.12). A higher percentage of cows ovulated in the immunized group than in the control group(91%(10/11) vs. 54%(7/13), respectively; P0.05). Moreover, inhibin-immunized cows had slightly larger corpus luteum(CL) than the controls 9 days after ovulation and significantly higher(P0.01) post-ovulation peak plasma progesterone(P4) concentrations. Immunization against inhibin also marginally increased the conception rate 42 days after insemination(45.8% vs. 15.4%; P0.05). These results demonstrate that immunization against inhibin, coupled with the treatment with the Ovsynch protocol, can constitute a new technique to increase fertility in water buffalo cows.     

Effects of Immunization Against Inhibin on Egg-Laying Performance in Magang and Landaise Geese

This study aimed to improve egg-laying performance in incubating Magang geese of Guangdong origin and Landaise geese of French origin. In experiment 1, 50 adults, egg-laying Magang geese were inoculated intramuscularly （i.m.） on days 0, 22, and 45 with 1 mL of immunogen containing 1 mg of recombinant chicken inhibin fusion protein. Immunization significantly increased blood antibody titers against inhibin fusion protein, but did not affect the egg-laying performance within 10 days after the first inoculation. From day 15, the egg-laying rate in inhibin-immunized group increased and was significantly higher than the values of control geese from day 40 to 55. However, the reverse was true from day 55 to 75 when more immunized geese developed incubation. In the entire 120 days of the experiment, the immunized geese laid 17.3 eggs in contrast to 16.4 eggs laid by the control geese. From day 30 till the end of the experiment, weight of eggs in the control geese was significantly greater than that in inhibin-immunized birds. In experiment 2, 40 Landaise geese were immunized against inhibin, as described in experiment 1. These geese laid 9.0 eggs on average in contrast to 7.3 eggs laid by nonimmunized control geese over 90 days of egg laying. The above results demonstrated that immunization against recombinant chicken inhibin fusion protein improved egg-laying performance in geese, and the increment was higher in nonincubating geese.     

Effect of Inhibin α(1-32)Gene Immunization on the Follicular Development and Reproductive Hormones in Rats

A total of 40 rats, aged in 30 days, were divided into 4 groups and immunized (intramuscularlyinjection) with 0 μg(control), 15 μg (group 1), 25 μg (group 2) or 40 μg(group 3) of inhibin α(1-32) re-combinant expression plasmid pcINH in combination with liposome. Booster was given without liposome onday 20 after primary immunization. The results showed that 50%(13/26) rats were detected in positive anti-body against inhibin. However, the increase of immunization dosage and booster did not promote the ratio ofantibody positive rats. The number of matured follicles above 0.8 mm in diameter in the antibody positive ratswas 2.3 more than that in the negative rats (P＞0.05). The concentration of blood plasma FSH increased dis-tinctively on day 10 after primary immunization (P＜0.05), but no increase was observed after booster immu-nization. The 17-β-estradiol levels in blood plasma of rats between the positive and the negative groups had noremarkable differences (P＞0.05). These results suggested that recombinant inhibin expression plasmid couldstimulate animal body to produce antibody against inhibin.     

抑制素主动免疫对青年奶山羊生殖的影响

本试验研究了抑制素主动免疫对青年奶山羊外周血生殖激素和产羔的影响。选用8只青年奶山羊,随机分成两组,每组各4只。试验组用人抑制素α亚单位片段(1-32)3次皮下注射进行免疫,生理盐水作对照,然后对8只羊间隔11d连续3次颈部肌肉注射氯前列烯醇(PG)0.1mg,末次注射PG发情后本交配种。结果为试验组4只奶山羊首免后均产生了抗体,末次免疫分别达到1∶64,1∶128,1∶64和1∶64,而对照组均未检测出抗体。8只青年奶山羊第3次注射PG后36～42h内全部发情,试验组的外周血促卵泡激素(FSH)的浓度高于对照组,且峰值的出现较对照组早,雌二醇(E2)的平均浓度显著高于对照组,而促黄体激素(LH)和孕酮(P)的平均浓度差异不显著。试验组比对照组产羔数提高80%。结果说明抑制素主动免疫能提高青年奶山羊的生殖性能。