| ObjectiveIn order to study whether long non-coding RNA is involved in the process of host immune cell differentiation,this paper has prepared animal model of antigen immunization –pathogen infection using rEg.P29 which has good immune protection effect on animals.On the basis of this,we study the differences in the expression of long non-coding RNA under the induction of rEg.P29 and pathogen infection.For exploring new ways to solve the problem of immunization interaction between host and pathogen and immune escape in parasitic diseases.Method1.Purification and preparation of rEg.P29.2.Preparation of antigen immunization –pathogen infection animal model: BALB / c mice were randomly divided into blank control group,P29 immunization +infection group and infection group.Mice in P29 immunization +infection group were immunized with rEg.P29(3 times),and mice infected with Echinococcus granulosus after three months of the third immunization interval(blank control group was not infected).Before the first immunization,2 weeks after the first immunization,2 weeks after the third immunization,the mice were sacrificed and the peripheral blood lymphocytes were prepared.6 months after infection,the mice in P29 immunization + infection group and infection group were killed.The number of cysts was counted and the immune protective effect of rEg.P29 was calculated.3.Total RNA was extracted: Total RNA was extracted from lymphocytes in peripheral blood of each group at each time point by TRIZOL method.4.Construction of lncRNA and mRNA libraries: Separation and construction of lncRNA using Epicentre’s Magnetic Core kit,and KAPA’s KAPA Strand mRNA-Seq Kit for isolation and construction.After purification with 2% agarose gel electrophoresis,the library was tested with Agilent 2100.5.High-throughput sequencing: The constructed lncRNA and mRNA libraries were sequenced using Illumina’s HiSeq 2000 high-throughput sequencer.high throughput sequencing: constructed lncRNA and mRNA libraries were sequenced by Illumina Solexa high throughput sequencing instrument.6.Bioinformatics methods to splice sequencing fragments: the tophat2 software was used to splice,the high quality reads was compared to the mouse genome,and the lncRNA and mRNA transcripts were constructed at different time points in each group.7.Bioinformatics method was used to screen the differentially expressed molecules: Cufflinks software was used to distribute all the reads,and the data could be compared with each other.Cuffdiff software was used to analyze the lncRNA and mRNA expression difference in experimental group and the control group according to P value≤0.05 and fold change≥2.8.Note: use lncRNAdb database(http://www.lncRNAdb.org/),UCSC database(http://genome.ucsc.edu/)to express the differential expression of lncRNA.9.Correlation analysis of lncRNA and corresponding mRNA: through the calculation of the experimental group and the control group at each time point between the expression of lncRNA and mRNA expression(reads number)of the Pearson coefficient(R),the absolute value of R is greater than the 0.99 threshold,to determine the relationship between lncRNA and mRNA.10.Preliminary validation of P29 immunization + infection group and control group after immunization with the differential expression of lncRNA and mRNA molecules: BALB/c mice were randomly divided into control group,immune group,mice were immunized by rEg.P29 for 3 times,the blank group did not,respectively,before the first immunization and two week after the third immunization,the eyeball blood preparation of peripheral blood lymphocytes.TRIZOL method was used to extract total RNA to construct cDNA library.Quantitative expression of differentially expressed lncRNA and related mRNA molecules by qPCR.Result1.The animal model was successfully constructed,and 94.8% protection can be induced by rEg.P292.The success of the extraction of total RNA,OD260/OD280 RNA of each group was between 1.8-2.0,more than 5ug.This results meet with the construction requirements.3.The mRNA library and lncRNA library were successfully constructed,through the Agilent 2100 test,meet with sequencing requirements.4.The results of mRNA library sequencing: 19717433 reads were obtained in the mRNA library of P29 immunization + infection group after immunization.Among them,the high quality reads was a total of 12375673,with a matching rate of 94.2%.24908555 reads were obtained in the mRNA library of P29 immunization + infection group after infection.Among them,the high quality reads was a total of 14766919,with a matching rate of 94.8%.25555287 reads were obtained in the mRNA library of blank control group after immunization.Among them,the high quality reads was a total of 15703026,with a matching rate of 96.1%.The blank control group received 23772436 reads after the infection,and the high quality reads were 15251560,the collation rate was 96.3%.In the infection group,the mRNA library received 21539925 reads,of which the high quality reads were 13748866 and the collation rate was 95.1%.5.The results of lncRNA library sequencing: 23367854 reads were obtained in the lncRNA library of P29 immunization + infection group after immunization.Among them,the high quality reads was a total of 16756424,with a matching rate of 85.1%.21803045 reads were obtained in the lncRNA library of P29 immunization + infection group after infection.Among them,the high quality reads was a total of 16449971,with a matching rate of 87.6 %.25501392 reads were obtained in the lncRNA library of blank control group after immunization.Among them,the high quality reads was a total of 18047705,with a matching rate of 84.7%.The blank control group received 21803045 reads after the infection,and the high quality reads were 16449971,the collation rate was 87.6%.In the infection group,the lncRNA library received 18171176 reads,of which the high quality reads were 12521894 and the collation rate was 95.1%.6.Screening of differentially expressed molecules: The different expressed molecules between P29 immunization + infection group and blank control group after immunization results: 17 lncRNAs,475 mRNAs.There were 14 different expressed lncRNAs and 85 mRNAs between the blank control group and infection group after infection.There were 30 different expressed lncRNAs and 192 mRNAs between the P29 immunization + infection group and infection group after infection.7.The correlation analysis results of lncRNA and the corresponding mRNA: the expression of lncRNA was significantly correlated with coding genes.8.Preliminary validation results: the differential expression of lncRNA and mRNA of P29 immunization + infection group and blank control group after immunization by qPCR validation.Ther were expression differences among lncRNA target molecules of XLOC-012763 and corresponding mRNA Dab2 ip,Hc,Trem,Oas3;INTE-015204 and corresponding mRNA Patz1,KLF4,Cxcl2 and NTE-028466 and corresponding mRNA Kler1,Jchain,results consistent with the correlation between the lncRNA and the corresponding mRNA analysis.Conclusion1.A total of 17 differentially expressed lncRNAs were obtained under the induction of vaccine,and mRNAs was found in a total of 473.A total of 14 differentially expressed lncRNAs were obtained under pathogen infection,and mRNAs was found in a total of 85.A total of 30 differentially expressed lncRNAs were obtained from vaccine induced pathogen infection,with a total of mRNA of 192.2.Initially identified lncRNA XLOC-012763 and corresponding mRNA Dab2 ip,Hc,Trem,Oas3;INTE-015204,and corresponding mRNA Patz1,KLF4,Cxcl2;INTE-028466 and corresponding mRNA Kler1,Jchain is the target lncRNA and target mRNA which related to obtain protection and may be involved in the induction of host immune response regulation,in order to solve the host pathogen of parasites the interaction between immune disease and immune escape mechanisms provide clues. |
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