The Distribution Of IgM And IgE Secretory Cells In The Palatine Tonsil Of Bactrian Camels

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.