猪FcRn在黏膜上皮中的表达与分布研究
摘要
黏膜是许多病原菌入侵机体的重要门户。一些组织器官可以通过它们的黏膜上皮来转运母源IgG,以使胎儿或新生儿获得被动免疫,而这个胞转过程,需要新生儿Fc受体(FcRn)的参与。研究表明,FcRn具有独特的重要功能,它不仅可以通过肠上皮、乳腺、胎盘和卵黄囊转运母源IgG,维持血液中IgG和白蛋白水平的相对稳定,还与动物的免疫应答密切相关。对FcRn的研究可以揭示机体转运IgG的机制,从而为兽医临床应用提供理论指导。
本研究通过分子生物学方法克隆表达了猪FcRn的胞浆尾区(FcRn-CT),并利用制备的兔抗猪FcRn-CT多克隆抗体,采用Western blot及免疫组织化学方法研究了FcRn在猪黏膜上皮中的表达及分布情况,从而为今后进一步开展猪黏膜上皮中表达的FcRn功能研究及应用提供理论依据。本研究取得的主要结果如下：
1.猪FcRn基因的克隆
根据GenBank上公布的猪FcRn基因序列设计引物,提取猪肝脏总RNA,利用RT-PCR方法扩增获得FcRn片段,回收后连接到pMD18-T载体上,对所得到的重组质粒pT-FcRn进行PCR、酶切鉴定及序列测定,与GenBank中登录号为AY740682的猪FcRn基因同源性为99%,进一步分析序列,结果显示FcRn的序列与AY740682的序列相比缺失81个碱基,即缺失27个氨基酸。
2.猪FcRn-CT的亚克隆及表达
以重组质粒pT-FcRn为模板,利用PCR方法亚克隆得到FcRn-CT基因片段,将目的基因分别连接到载体pGEX-KG和pET-32a(+)上构建重组原核表达质粒(KG-CT和pET-32a-CT),并成功在大肠杆菌中获得了表达,对表达产物进行SDS-PAGE电泳分析,结果显示分别出现了分子量大小为29 KDa和23 KDa的蛋白条带,与预期融合蛋白GST-CT和His-CT的分子量大小相符。
3.兔抗猪FcRn多克隆抗体的制备与纯化
利用纯化的融合蛋白GST-CT作为免疫原,His-CT作为检测原,按照设计好的免疫程序免疫家兔,成功制备了兔抗猪FcRn高免血清,用间接ELISA法检测血清抗体效价高达1：32000,并采用亲和层析法对所获得的高免血清进行了纯化。
4.初步应用Western blot方法检测猪FcRn在黏膜上皮中的表达
利用制备的兔抗猪FcRn高免血清,用Western blot方法检测猪FcRn在黏膜上皮中的表达情况。结果显示,在猪气管、肺、十二指肠、子宫、阴道等黏膜上皮中均有FcRn表达。
5.应用免疫组织化学方法检测猪FcRn在黏膜上皮中的表达与分布
利用纯化的兔抗猪FcRn多克隆抗体,建立了猪FcRn的免疫组织化学检测方法。结果显示,在猪子宫、阴道、鼻腔、气管、肺、十二指肠、空肠中均表达,且主要定位于子宫内膜上皮、阴道黏膜上皮、鼻腔前庭部和呼吸部黏膜上皮、气管黏膜上皮和气管腺、肺泡和细支气管黏膜上皮、十二指肠黏膜上皮、空肠黏膜上皮等部位。
The mucosal surface of the body is an important gateway for many pathogenic bacteria invasion. The immunoglobulin IgG can be transported across their epithelial surfaces in several organs, in order to obtain passive immunity from the mother to the fetus or newborn. The neonatal Fc receptor (FcRn) plays an important role in this process. Researchers have shown that FcRn performs unique and important functions, which not only plays a critical role in IgG transcytosis across intestinal epithelium, mammary gland, placenta and yolk sac from mother to baby, mentain the homeostasis of serum IgG and albumin proteins as well, and it is also closely related with animal immune response. Research on FcRn can help to reveal IgG transfer mechanism and thus provides theoretical guidance for veterinary clinical application.
The cytoplasma tail of porcine FcRn (FcRn-CT) was subcloned and expressed in E.coli by molecular biology, the specific rabbit anti-porcine FcRn polyclonal antibody was prepared, and the expression and localization of FcRn protein in porcine mucous epithelium was analysed by western blot and immunohistochemistry. These results will provide the foundation for further research on functions and applications of FcRn in mucous epithelium in the future. The research contents are summarized as follows:
1. Cloning of procine FcRn gene
According to the Sus scrofa FcRn mRNA sequence published in GenBank, FcRn gene was amplified from total RNA of porcine liver by RT-PCR and cloned into the pMD18-T. The recombinant plasmids were identified by PCR, restriction enzyme analysis and sequencing. The sequence analysis demonstrated that the FcRn gene had 99% homology with AY740682, and there is a deletion of 27 amino acids compared with AY740682.
2. Subcloning and expression of procine FcRn-CT gene
FcRn-CT gene were amplified from the recombinant plasmid pT-FcRn by PCR and cloned into the prokaryotic expression vector pGEX-KG and pET-32a(+).The recombinant plasmid was transformed into E. coli BL21 and successfully expressed. The expressed GST-CT and His-CT fusion protein in E. coli BL21 is about 29 KDa,23 KDa in the analysis of SDS-PAGE, respectively.
3. Preparation and purification of rabbit anti-porcine FcRn polyclonal antibody
Rabbits were immunized with purified GST-CT fusion protein which was emulsified with Frund's adjuvant, the titer of the antiserum against porcine FcRn was as high as 1:32000 detected by indirect ELISA assay using purified His-CT fusion protein.
4. Expression of FcRn protein in porcine mucous epithelium was detected by Western blot Western blot analysis for detecting FcRn on porcine mucous epithelium was established by using rabbit anti-porcine FcRn-CT serum.The results indicated that FcRn was expressed in the tissues of trachea, lung, duodenum, uterus and vagina.
5. Expression and distribution of FcRn protein in porcine mucous epithelium was detected by immunohistochemistry
Immunohistochemistry analysis for detecting FcRn on porcine mucous epithelium was established by using purified rabbit anti-porcine FcRn-CT polyclonal antibody. The results showed that FcRn was expressed in the tissues of uterus, vagina, nasal cavity, trachea, lung, duodenum and jejunum, and the positive signals were in the uterine endometrial epithelium, vaginal epithelium, nasal vestibular mucosa epithelium and respiratory mucous epithelium, trachea mucosa and tracheal gland, duodenal mucosa, jejunum epithelium.
本研究通过分子生物学方法克隆表达了猪FcRn的胞浆尾区(FcRn-CT),并利用制备的兔抗猪FcRn-CT多克隆抗体,采用Western blot及免疫组织化学方法研究了FcRn在猪黏膜上皮中的表达及分布情况,从而为今后进一步开展猪黏膜上皮中表达的FcRn功能研究及应用提供理论依据。本研究取得的主要结果如下：
1.猪FcRn基因的克隆
根据GenBank上公布的猪FcRn基因序列设计引物,提取猪肝脏总RNA,利用RT-PCR方法扩增获得FcRn片段,回收后连接到pMD18-T载体上,对所得到的重组质粒pT-FcRn进行PCR、酶切鉴定及序列测定,与GenBank中登录号为AY740682的猪FcRn基因同源性为99%,进一步分析序列,结果显示FcRn的序列与AY740682的序列相比缺失81个碱基,即缺失27个氨基酸。
2.猪FcRn-CT的亚克隆及表达
以重组质粒pT-FcRn为模板,利用PCR方法亚克隆得到FcRn-CT基因片段,将目的基因分别连接到载体pGEX-KG和pET-32a(+)上构建重组原核表达质粒(KG-CT和pET-32a-CT),并成功在大肠杆菌中获得了表达,对表达产物进行SDS-PAGE电泳分析,结果显示分别出现了分子量大小为29 KDa和23 KDa的蛋白条带,与预期融合蛋白GST-CT和His-CT的分子量大小相符。
3.兔抗猪FcRn多克隆抗体的制备与纯化
利用纯化的融合蛋白GST-CT作为免疫原,His-CT作为检测原,按照设计好的免疫程序免疫家兔,成功制备了兔抗猪FcRn高免血清,用间接ELISA法检测血清抗体效价高达1：32000,并采用亲和层析法对所获得的高免血清进行了纯化。
4.初步应用Western blot方法检测猪FcRn在黏膜上皮中的表达
利用制备的兔抗猪FcRn高免血清,用Western blot方法检测猪FcRn在黏膜上皮中的表达情况。结果显示,在猪气管、肺、十二指肠、子宫、阴道等黏膜上皮中均有FcRn表达。
5.应用免疫组织化学方法检测猪FcRn在黏膜上皮中的表达与分布
利用纯化的兔抗猪FcRn多克隆抗体,建立了猪FcRn的免疫组织化学检测方法。结果显示,在猪子宫、阴道、鼻腔、气管、肺、十二指肠、空肠中均表达,且主要定位于子宫内膜上皮、阴道黏膜上皮、鼻腔前庭部和呼吸部黏膜上皮、气管黏膜上皮和气管腺、肺泡和细支气管黏膜上皮、十二指肠黏膜上皮、空肠黏膜上皮等部位。
The mucosal surface of the body is an important gateway for many pathogenic bacteria invasion. The immunoglobulin IgG can be transported across their epithelial surfaces in several organs, in order to obtain passive immunity from the mother to the fetus or newborn. The neonatal Fc receptor (FcRn) plays an important role in this process. Researchers have shown that FcRn performs unique and important functions, which not only plays a critical role in IgG transcytosis across intestinal epithelium, mammary gland, placenta and yolk sac from mother to baby, mentain the homeostasis of serum IgG and albumin proteins as well, and it is also closely related with animal immune response. Research on FcRn can help to reveal IgG transfer mechanism and thus provides theoretical guidance for veterinary clinical application.
The cytoplasma tail of porcine FcRn (FcRn-CT) was subcloned and expressed in E.coli by molecular biology, the specific rabbit anti-porcine FcRn polyclonal antibody was prepared, and the expression and localization of FcRn protein in porcine mucous epithelium was analysed by western blot and immunohistochemistry. These results will provide the foundation for further research on functions and applications of FcRn in mucous epithelium in the future. The research contents are summarized as follows:
1. Cloning of procine FcRn gene
According to the Sus scrofa FcRn mRNA sequence published in GenBank, FcRn gene was amplified from total RNA of porcine liver by RT-PCR and cloned into the pMD18-T. The recombinant plasmids were identified by PCR, restriction enzyme analysis and sequencing. The sequence analysis demonstrated that the FcRn gene had 99% homology with AY740682, and there is a deletion of 27 amino acids compared with AY740682.
2. Subcloning and expression of procine FcRn-CT gene
FcRn-CT gene were amplified from the recombinant plasmid pT-FcRn by PCR and cloned into the prokaryotic expression vector pGEX-KG and pET-32a(+).The recombinant plasmid was transformed into E. coli BL21 and successfully expressed. The expressed GST-CT and His-CT fusion protein in E. coli BL21 is about 29 KDa,23 KDa in the analysis of SDS-PAGE, respectively.
3. Preparation and purification of rabbit anti-porcine FcRn polyclonal antibody
Rabbits were immunized with purified GST-CT fusion protein which was emulsified with Frund's adjuvant, the titer of the antiserum against porcine FcRn was as high as 1:32000 detected by indirect ELISA assay using purified His-CT fusion protein.
4. Expression of FcRn protein in porcine mucous epithelium was detected by Western blot Western blot analysis for detecting FcRn on porcine mucous epithelium was established by using rabbit anti-porcine FcRn-CT serum.The results indicated that FcRn was expressed in the tissues of trachea, lung, duodenum, uterus and vagina.
5. Expression and distribution of FcRn protein in porcine mucous epithelium was detected by immunohistochemistry
Immunohistochemistry analysis for detecting FcRn on porcine mucous epithelium was established by using purified rabbit anti-porcine FcRn-CT polyclonal antibody. The results showed that FcRn was expressed in the tissues of uterus, vagina, nasal cavity, trachea, lung, duodenum and jejunum, and the positive signals were in the uterine endometrial epithelium, vaginal epithelium, nasal vestibular mucosa epithelium and respiratory mucous epithelium, trachea mucosa and tracheal gland, duodenal mucosa, jejunum epithelium.
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