CN110408623A - 一种提高鹅产蛋性能的方法 - Google Patents
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Abstract
本发明公开了一种提高鹅产蛋性能的方法,包括以下步骤:(1)从鹅的卵巢组织提取总RNA并扩增出鹅INH‑α基因CDS区序列,插入原核表达载体PET‑30a(+)载体NdeI/HindIII酶切位点中;(2)将上述载体转化重组产物,构建重组表达质粒;(3)将上述重组表达质粒转化到大肠杆菌中培养,诱导,收集菌体,采用超声波破碎后,离心得含有蛋白的包涵体;(4)从上述包涵体中分离纯化蛋白,得重组融合蛋白;(5)将纯化的重组融合蛋白制成疫苗,免疫产蛋鹅。相对于现有技术,本发明方法能够显著提高产蛋鹅体内小卵泡数量和产蛋量,提高鹅的产蛋性能,且简单易行,为解决鹅的产蛋量低的技术瓶颈提供新方法,为鹅产业发展提供了有效技术支持,具有良好的应用前景免疫产蛋鹅。
Description
技术领域
本发明涉及一种提高鹅产蛋性能的方法,属于鹅产蛋技术领域。
背景技术
中国是鹅养殖和消费大国,养殖量占世界总量的93.2%,占据绝对地位。但鹅产蛋性能低下是制约鹅产业发展的关键瓶颈。鹅产蛋量性状遗传力低,通过常规选育来提高鹅的产量性能难度较大。一些科学家也试图通过饲料优化和环境控制等技术手段来提高鹅的产蛋性能。如赵鑫和龙英孜等通改变过维生素、大豆黄酮和能量蛋白水平以及补料方式来提高提高鹅的产蛋(维生素、大豆黄酮和能量蛋白水平对浙东白鹅母鹅繁殖性能的影响,赵鑫,南京农业大学硕士学位论文,2012;三招补料提高母鹅产蛋率,龙英孜等,农家科技,2016(17):38-38)。霍明东和陈永华等调控光照时间和光照周期来提高鹅的产蛋(如何提高母鹅产蛋量,霍明东等,水禽世界,2011,(1):18-19;通过光照周期的调控来提高鹅的产蛋性能,陈永华等,水禽世界,2011,(2):41-44);这些方法仅能一定程度上提高鹅的产蛋性能,而且耗时费力。
因此,目前提高鹅产蛋性能一般通过饲料优化,控制环境等技术措施,但这些方法仅能一定程度上提高鹅的产蛋性能,而且耗时费力。
发明内容
发明目的:针对上述技术问题,本发明提供了一种提高鹅产蛋性能的方法,其是利用INH-α重组蛋白免疫中和作用。即采用INH-α重组蛋白主动免疫具有抑制产蛋作用基因INH-α,使浙东白鹅体内产生相应的抗体,从而减弱其体内INH-α的抑制作用,最终提升鹅产蛋量。
技术方案:为了达到上述发明目的,本发明所采用的技术方案如下:
一种提高鹅产蛋性能的方法,包括以下步骤:
(1)从鹅的卵巢组织提取总RNA并扩增出鹅INH-α基因CDS区序列,插入原核表达载体PET-30a(+)载体NdeI/HindIII酶切位点中;
(2)将上述载体转化重组产物,构建重组表达质粒;
(3)将上述重组表达质粒转化到大肠杆菌中培养,诱导,收集菌体,采用超声波破碎后,离心得含有蛋白的包涵体;
(4)从上述包涵体中分离纯化蛋白,得重组融合蛋白;
(5)将纯化的重组融合蛋白制成疫苗,免疫产蛋鹅。
作为优选:
步骤(1)中采用TRIzol法从鹅的卵巢组织提取总RNA,并采用PCR扩增出鹅INH-α基因CDS区序列。
步骤(3)中大肠杆菌中选自大肠杆菌E.coli BL21。
步骤(3)中用酶标仪检测培养菌液的吸光度值(OD),当OD600=0.6-0.8时,进行诱导。
步骤(3)中采用IPTG进行诱导,优选0.2mM IPTG,15℃诱导16h。
步骤(4)中采用Ni-IDA亲和层析分离纯化蛋白,收集纯化的蛋白进行透析,复性,过滤,即得所述重组融合蛋白。
步骤(5)中所述疫苗选自油乳剂疫苗,免疫方式为肌肉注射。
步骤(5)中免疫产蛋鹅的方法为:每隔10天免疫1次,共免疫2-3次
抑制素(INH)通过反馈调节机制抑制(FSH)的合成与分泌。针对这点,本发明提出一种新的激素免疫的方法,即通过主动免疫INH-α重组蛋白,诱导鹅体内产生相应抗体,降低家禽机体内源性卵泡抑制素的生物活性,导致机体循环中卵泡刺激激素(FSH)浓度升高,使排卵率和产蛋率增加,最终提高鹅的产蛋性能。
技术效果:相对于现有技术,本发明方法能够显著提高产蛋鹅体内小卵泡数量和产蛋量,提高鹅的产蛋性能,且简单易行,为解决鹅的产蛋量低的技术瓶颈提供新方法,为鹅产业发展提供了有效技术支持,具有良好的应用前景。
附图说明
图1为INH-α基因CDS区扩增结果;
图2为INH-α基因原核表达载体质粒(Lane1)及双酶切(Lane 2)鉴定;
图3为包涵体中蛋白纯化结果;
图4为Western Blot检测重组菌表达融合蛋白。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明。
实施例
(1)取就巢期浙东白鹅卵巢组织。采用TRIzol法提取卵巢总RNA,并合成cDNA。
(2)采用PCR扩增INH-α基因CDS区,引物信息如下:
F:TGTTTAACTTTAAGAAGGAGATATACATATGCACCACCATCACCATCATCATTTTGGCCTGTGCCTGCATCACGGTCAAG;
R:TAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTCATTAACCTTGCCCCA)
PCR结果如图1所示,纯化PCR产物,插入PET-30a(+)载体NdeI/HindIII酶切位点中,双酶切鉴定,双酶切结果如图2所示,酶切位点为NdeI/HindIII。
(3)将构建好的重组片段转化重组产物,构建重组表达质粒。
(4)将所述重组表达质粒转化到大肠杆菌E.coli BL21(购于Novagen公司)中,生长至OD600=0.6-0.8时,在培养液中加入终浓度0.2mM的IPTG于15℃诱导16小时,收集菌体,采用超声波破碎后,离心得含有蛋白的包涵体。
(5)采用50mM Tris(pH=8.0),300mM NaCl含1%TritonX-100,2mM EDTA,5mM DTT洗涤,以50mM Tris(pH=8.0),300mM NaCl,20mM Imidazole,8M Urea缓冲液溶解包涵体,同时平衡Ni-IDA柱,最后用不同浓度咪唑的平衡缓冲液洗脱目标蛋白,并收集每个洗脱组分进行SDS-PAGE分析检测。经Ni-IDA亲和层析纯化分析,收集纯度相对较高的泳道,将其加入到处理后的透析袋中,4℃环境下,透析到缓冲液[1×PBS(pH8.0),4mM GSH,0.4mM GSSG,0.4M L-Arginine,1M Urea]中复性,复性后蛋白最终透析于储存液1×PBS(pH=8.0)溶液约6-8h。透析复性结束后,上清用0.22μm滤器过滤后分装并测定浓度,最终将其冻存至-80℃。
包涵体溶解后运用SDS-PAGE检测包涵体中蛋白纯化结果,结果如图3所示。
(6)取一管蛋白进行冻融实验,并采用Western Blot检测重组菌表达融合蛋白,经Western Blot检测INH-α重组蛋白大小约为72kDa,结果如图4所示。
(7)采用现配现用的原则,将蛋白与免疫佐剂进行1∶1混合,采用双注射器反复推拉的方式,制备油乳剂。第一次与完全免疫佐剂混合,第二次与不完全免疫佐剂混合。
(8)选取处于产蛋前期的浙东白鹅,母鹅胸肌处多点注射免疫乳化疫苗(每只0.8mg蛋白),间隔10天,再次免疫。
(9)经过上述主动免疫,浙东白鹅体内小卵泡数量和产蛋性能明显提高。表1列举了主动免疫INH-α重组蛋白效果,结果如下:
上表1数据显示,采用本发明方法后,产蛋鹅体内小卵泡数量可提高1.6倍,产蛋量提高25%,方法简单易行,为解决鹅的产蛋量低的技术瓶颈提供新方法。
Claims (8)
1.一种提高鹅产蛋性能的方法,其特征在于,包括以下步骤:
(1)从鹅的卵巢组织提取总RNA并扩增出鹅INH-α基因CDS区序列,插入原核表达载体PET-30a(+)载体NdeI/HindIII酶切位点中;
(2)将上述载体转化重组产物,构建重组表达质粒;
(3)将上述重组表达质粒转化到大肠杆菌中培养,诱导,收集菌体,采用超声波破碎后,离心得含有蛋白的包涵体;
(4)从上述包涵体中分离纯化蛋白,得重组融合蛋白;
(5)将纯化的重组融合蛋白制成疫苗,免疫产蛋鹅。
2.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(1)中采用TRIzol法从鹅的卵巢组织提取总RNA,并采用PCR扩增出鹅INH-α基因CDS区序列。
3.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(3)中大肠杆菌中选自大肠杆菌E.coli BL21。
4.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(3)中用酶标仪检测培养菌液的吸光度值(OD),当OD600=0.6-0.8时,进行诱导。
5.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(3)中采用IPTG进行诱导。
6.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(4)中采用Ni-IDA亲和层析分离纯化蛋白,收集纯化的蛋白进行透析,复性,过滤,即得所述重组融合蛋白。
7.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(5)中所述疫苗选自油乳剂疫苗,免疫方式为肌肉注射。
8.根据权利要求1所述的提高鹅产蛋性能的方法,其特征在于,步骤(5)中免疫产蛋鹅的方法为:每隔10天免疫1次,共免疫2-3次。
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