【作者】 李敏；

【导师】 王雯慧；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2019， 硕士

【摘要】 双峰驼是我国西北部地区特色动物,主要生存于荒漠地带,由于其分布地区人烟稀少,经常不被人们所重视,目前国内对双峰驼的研究较少,对双峰驼的IgM与IgE更是少有人研究。咽部是饮食和呼吸气的必经之路,经常接触病原体,因此机体为适应环境,而在此产生了丰富的淋巴组织保护口咽部,腭扁桃体是其中最大的淋巴组织。为明确IgM~+与IgE~+分泌细胞在双峰驼腭扁桃体内的分布特点及其与黏膜免疫的关系,本实验分为双峰驼IgM和IgE的原核表达、多克隆抗体的制备及运用免疫组织化学技术确定IgM~+与IgE~+浆细胞在腭扁桃体的分布三个部分:(1)为获取双峰驼IgM与IgE表达产物及多克隆抗体,首先找出GenBank收录的双峰驼IgM、IgA、IgE、IgG重链恒定区的氨基酸序列,通过比对双峰驼IgM与双峰驼IgA、IgE、IgG的氨基酸序列,选取一段差异明显的氨基酸序列,将氨基酸序列转化为对应的基因片段,对IgE也采取相同方法选取出对应基因片段。人工合成目的基因片段,PCR扩增目的基因片段,插入pET-28a(+)载体构建重组载体,转化进大肠杆菌BL21,并诱导重组蛋白表达,SDS-PAGE鉴定重组蛋白。结果显示,成功构建了表达载体pET-28a-IgM与pET-28a-IgE,诱导表达并纯化出目的蛋白经鉴定也与预期相符。(2)纯化的目的蛋白分别免疫家兔制备抗血清,ELISA和Western Blot鉴定获得的血清。IgM的抗血清用ELISA方法检测,效价为1∶32000,Western Blot结果显示,IgM的兔抗血清具有良好的反应原性。IgE的抗血清用ELISA方法检测,效价为1∶16000,Western Blot结果显示,IgE的兔抗血清也具有良好的反应原性。(3)根据SABC的显色结果,将IgM~+与IgE~+分泌细胞在双峰驼腭扁桃内的分布情况分为上皮下区、网状上皮下区、滤泡区和滤泡间区。另外发现IgM~+分泌细胞在双峰驼腭扁桃体内上皮下区、网状上皮下区、滤泡区和滤泡间区都有存在,在网状上皮下区和滤泡区分布密集,且数量多,而在滤泡间区和上皮下区呈散在分布,数量较少。对IgM~+分泌细胞分布密度进行数据统计及分析得出,IgM~+分泌细胞在网状上皮下区数量显著高于滤泡间区和上皮下区(P<0.05)。滤泡间区与上皮下区的差异不显著(P>0.05)。IgE~+分泌细胞分布于上皮下区、网状上皮下区及滤泡间区,在网状上皮下区分布相对密集,数量较多,上皮下区和滤泡间区中IgE~+分泌细胞偶见。上述结果证实了,双峰驼IgM与IgE的重组蛋白成功表达及多克隆抗体的成功制备,SABC显色结果也证明了制备的兔抗血清能很好地与天然蛋白相结合,双峰驼IgM和IgE分泌细胞在腭扁桃体内的分布部位表明其具有在该些部位行使黏膜免疫功能的可能,并为之提供条件。更多还原

【Abstract】 Bactrian camel is a characteristic animal in the northwest part of China.It lives in the desert area.Because its distribute of sparsely populated areas,it is often not valued by people.At present,there are few studies on Bactrian camel in China,and IgM and IgE on Bactrian camels are less.The pharynx is the only way to eat and breathe.It is often exposed to pathogens.Therefore,the body adapts to the environment,and it produces abundant lymphoid tissue to protect the oropharynx,The tonsil is the largest lymphoid tissue.In order to clarify the distribution characteristics of IgM~+and IgE~+secretory cells in the Bactrian camel’s amygdala and the subsequent study on the mucosal immune system of Bactrian camel,this experiment is divided into prokaryotic expression of Bactrian camel IgM and IgE,preparation of polyclonal antibody.And using immunohistochemical techniques to determine the distribution of IgM~+and IgE~+plasma cells in the tonsil:(1)To obtain the expression products and antibodies of the heavy chain constant region of IgM and IgE in Bactrian camel,firstly find the amino acid sequence of the heavy chain constant region of IgM,IgA,IgE and IgG contained in GenBank,and compare the double peaks.The amino acid sequences of Camel IgM and Bactrian camel IgA,IgE and IgG were selected,and a large amino acid sequence was selected to convert the amino acid sequence into the corresponding gene fragment.The corresponding gene fragment was also selected by the same method for IgE.Then,the selected gene fragment was codon-optimized according to the codon preference of E.coli,and the target gene fragment was amplified by PCR,inserted into pET-28a(+)vector to construct a recombinant vector,transformed into E.coli BL21,and induced to recombine.Protein expression,SDS-PAGE identified recombinant protein.The results showed that the expression vectors pET-28a-IgM and pET-28a-IgE were successfully constructed,and the expression and purification of the target protein were also confirmed.(2)The purified target protein was used to immunize rabbits to prepare antiserum,and the obtained serum was identified by ELISA and Western Blot.The antiserum of IgM was detected by ELISA,and the titer was 1:32000.The results of Western Blot showed that the rabbit antiserum of IgM had good reactogenicity.The antiserum of IgE was detected by ELISA,and the titer was 1:16000.The results of Western Blot showed that the rabbit antiserum of IgE also had good reactogenicity.(3)According to the results of immunohistochemical technique,the distribution of IgM~+and IgE~+secreting cells in the Bactrian camel’s almond was divided into subepithelial area,reticular subepithelial area,follicular area and follicular area.In addition,IgM~+secreting cells were found to exist in the subepithelial region,the reticular epithelial region,the follicular region and the follicular region of the Bactrian camel.The secretory cells of IgM~+are densely distributed in the subretinal and follicular regions of the crypt,and the number is large.It is scattered in the interfollicular and subepithelial regions,and the number is small.Statistical analysis of the distribution of IgM~+secreting cells showed that the number of IgM~+secreting cells in the reticular subepithelial area was significantly higher than that in the interfollicular area and subepithelial area(P<0.05).There was no significant difference between the interfollicular area and the subepithelial area(P>0.05).The IgE~+secretory cells are distributed in the subepithelial area,the reticular subepithelial area and the follicular area,and the distribution in the reticular subepithelial area is relatively dense and the number is large.IgE~+secreting cells are occasionally seen in the subepithelial region,and there are few IgE~+secreting cells in the interfollicular region,which are scattered.The above results confirmed the successful expression of the recombinant protein of Bactrian camel IgM and IgE and the successful preparation of its polyclonal antibody.The results of immunohistochemistry also confirmed that the prepared antiserum can be well combined with the native protein,The distribution of Bactrian camel IgM and IgE secreting cells in the amygdala indicates that it has the potential to exert mucosal immune function at these sites and provide conditions for it.更多还原