CN114507726A

CN114507726A - Screening method of toxoplasma infection animal host brain tissue differential expression gene and application thereof

Info

Publication number: CN114507726A
Application number: CN202210063595.XA
Authority: CN
Inventors: 袁浩; 袁子国; 张锡锐; 张秀香; 杨子鹏; 闫紫荆; 王艳云; 张翩; 任照文; 宋伊宁
Original assignee: South China Agricultural University; Xinjiang Agricultural University
Current assignee: South China Agricultural University; Xinjiang Agricultural University
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-05-17

Abstract

The invention provides a screening method of differential expression genes of a brain tissue of a toxoplasma infected animal host and application thereof, wherein the differential expression genes of the brain tissue of the toxoplasma infected animal host are screened and identified by analyzing the gene expression differential condition of the brain tissue of the animal host before infection of the toxoplasma through the application of a transcriptome sequencing technology in cooperation with bioinformatics. The method can quickly and effectively research gene expression difference, provides reference for revealing the body immune response of the animal host infected with the toxoplasma, verifies 10 differentially expressed genes CCL5, CCL8, CCL23, CASP8, TLR1, TLR4, STAT1, ROS1, NOS1 and NOS2 by adopting a qRT-PCR method, takes the screened differentially expressed genes as target genes for diagnosing the brain infection of the host toxoplasma, is favorable for accurately distinguishing the brain toxoplasma infection of the animal host including fruit raccoon and other wild animals, and provides a diagnosis target for diagnosing the toxoplasma infection of the wild animals.

Description

Screening method of toxoplasma infection animal host brain tissue differential expression gene and application thereof

Technical Field

The invention relates to the field of parasitology, in particular to a screening method of genes differentially expressed in brain tissues of toxoplasma infected animal hosts and application thereof.

Background

Toxoplasma is a protozoan that is obligately parasitic within eukaryotic cells. Toxoplasmosis caused by toxoplasma infection is an important zoonosis, which can cause serious clinical symptoms and even death of infants, pregnant women and people with low immune function, and one of the main ways of causing toxoplasmosis is that people eat food or water containing toxoplasmosis oocysts by mistake. Toxoplasma has three infectious stages: sporozoites, tachyzoites and bradyzoites. Toxoplasma tachyzoites can invade almost all nucleated cells extensively, and metaneurotic cells can cause Toxoplasma encephalitis. Epidemiological investigations have shown that about 30% of the world population has a potential toxoplasma infection, of which 90% of patients with toxoplasma encephalitis die, with a higher proportion of secondary paralysis.

Felines are the only final hosts for toxoplasma. However, toxoplasma gondii can have many intermediate hosts, including almost all mammals, fish and birds, while humans are also one of the important intermediate hosts for toxoplasma gondii. Toxoplasma gondii can accomplish sexual and apomictic reproduction in both the terminal and intermediate hosts. The process of sexual reproduction is mainly completed in a feline body, after the feline inhales cysts, pseudocysts or oocysts, the toxoplasma gondii at each stage can invade into the small intestine of the feline to start to proliferate, after multiple times of division and proliferation, male and female gametophytes can be formed, and then become male and female gametophytes in a certain stage, after fertilization, zygotes are formed, the zygotes are developed into oocysts, and the oocysts are finally discharged out of the body along with the excrement of the domestic cat, so that the process of sexual reproduction is completed. Paguma larvata belongs to the family of civets of the order carnivora and is distributed in 62 vast areas in the south of the yellow river in China. Are important carriers of parasites, bacteria and viruses. It has been reported that zoonotic pathogens including toxoplasma gondii, enterozoonosis, giardia duodenalis, salmonella enteritidis, campylobacter and cryptosporidium are carried and transmitted by paguma larvata.

With the advent of the post-genome era, various omics technologies such as transcriptomics, proteomics, metabolomics and the like are developed successively, wherein the transcriptomics is the most mature technology currently developed. Transcriptomics are studies of the type and copy number of mrnas contained in a living cell in a certain functional state, reflecting the expression of all genes in the cell at a certain time and space. The genetic center rule suggests that genetic information is transferred from DNA to protein via mRNA. Therefore, mRNA is important for the study of DNA and protein as a "bridge" for information transfer.

At present, the researches on wild animals such as Toxoplasma gondii infected masked palm civet are mainly focused on the epidemiology, Toxoplasma gondii antibodies are detected by a serological method, Toxoplasma gondii DNA is detected in brain tissues by a PCR method, and the damage of tissues and cell levels caused by Toxoplasma gondii infection is observed by making pathological tissue sections, but no report is made on a method for researching the differential expression genes of the brain tissues of the Toxoplasma gondii infected masked palm civet by applying a transcriptome sequencing technology.

Disclosure of Invention

In order to solve the problems, the invention provides a method for researching a differential expression gene of a toxoplasma chronic masked-civet brain tissue by applying an RNA-seq technology, a transcriptome analysis method of a mechanism of toxoplasma ME49 strain infection-induced masked-civet brain tissue damage and application thereof.

The invention provides a screening method of genes differentially expressed by brain tissues of toxoplasma infected animal hosts, which comprises the following steps:

(1) extracting animal host brain tissue RNA, constructing a library and sequencing transcriptome;

(2) filtering sequencing original data, removing low-quality reads with a connector, ploy-N and sequencing primers, and calculating sequence repeatability of Q20, Q30, GC content and clean reads to obtain high-quality clean reads;

(3) splicing clean reads by using a Trinity program to obtain a transcript sequence, and taking the longest transcript in each gene as a Unigene;

(4) comparing clean reads to databases SWISS-PROT, NR, KEGG, KOG and Pfam for gene function annotation;

(5) the gene expression level was evaluated by calculating the FPKM value of each gene using the MARS model in the DEGseq program, high expression genes with FPKM values greater than 1000, setting P<0.05，|log₂ ^{(fold change)}| ≧ 1, FDR-corrected q-value<0.05, marking the difference significant threshold value meeting the conditions as significan, and screening the significant difference expression genes;

(6) setting a standard of significance screening: p is less than 0.05, and gene functional classification is carried out on the significant difference expression genes through a GO database;

(7) setting a standard of significance screening: p <0.05, pathway analysis was performed on significantly differentially expressed genes by KEGG database.

In one embodiment of the present invention, the significantly differentially expressed genes selected in step (5) are not less than 2808 genes, wherein the genes with up-regulated expression are not less than 860 genes, and the genes with down-regulated expression are not less than 1948 genes.

In one embodiment of the present invention, step (6) obtains genes with significant difference annotated as specific GO no less than 2808 genes, wherein significant GO term resulting in up-regulated difference genes is no less than 860 and significant GO term resulting in down-regulated difference genes is no less than 1948.

In one embodiment of the invention, the genes with significant differences annotated as specific KEGG obtained in step (7) are 933 genes, significantly enriched into 100 pathways, and the first 3 pathways with the most differentially expressed genes are Jak-STAT signaling pathway (28), chemokine signaling pathway (23) and Toll-like receptor signaling pathway (19), respectively.

In a second aspect, the present invention provides a target site for detecting toxoplasma infection, wherein the target site is a gene differentially expressed in brain tissue of a host infected with toxoplasma, and the target site comprises:

1) one or more of transcriptional up-regulation genes CCL5, CCL8, CCL23, CASP8, TLR1, TLR 4;

and/or

2) Transcription down-regulation of one or more of the genes STAT1, ROS1, NOS1, NOS 2.

In a third aspect, the invention provides a primer for detecting Toxoplasma gondii infection, said primer being designed for a target site according to the second aspect of the invention, comprising one or more of the following sets of primers 1) to 10):

a primer set for CCL5 as shown in SEQ ID NO. 1-2;

2) a primer set for CCL8 as set forth in SEQ ID nos. 3-4;

3) a primer set for CCL23 as set forth in SEQ ID nos. 5-6;

4) primer sets for CASP8 as set forth in SEQ ID nos. 7-8;

5) a primer group for TLR1 as shown in SEQ ID NO. 9-10;

6) a primer set for TLR4 shown as SEQ ID NO. 11-12;

7) a primer set for STAT1 as set forth in SEQ ID nos. 13-14;

8) a primer set for ROS1 as shown in SEQ ID NO. 15-16;

9) primer sets for NOS1 as shown in SEQ ID NO. 17-18;

10) primer sets for NOS2 as shown in SEQ ID NO. 19-20.

In a fourth aspect, the invention provides a kit for detecting Toxoplasma gondii infection, comprising the primers of the third aspect of the invention.

The fifth aspect of the present invention provides a detection method for detecting Toxoplasma gondii infection, which uses the differential expression gene of brain tissue after the Toxoplasma gondii infection host according to the second aspect of the present invention as a detection object;

when the transcription of one or more genes in CCL5, CCL8, CCL23, CASP8, TLR1 and TLR4 is up-regulated, and/or the transcription of one or more genes in STAT1, ROS1, NOS1 and NOS2 is down-regulated, the brain of the host is judged to be infected with Toxoplasma gondii.

In one embodiment of the present invention, the detection method uses the primer according to the third aspect of the present invention to detect the host brain tissue.

In a sixth aspect, the invention provides a screening method for genes differentially expressed in brain tissue of a toxoplasma gondii infected animal host according to the first aspect of the invention, a target site for detecting toxoplasma gondii infection according to the second aspect of the invention, a primer for detecting toxoplasma gondii infection according to the third aspect of the invention, a kit for detecting toxoplasma gondii infection according to the fourth aspect of the invention or a detection method for detecting toxoplasma gondii infection according to the fifth aspect of the invention, and the application of the screening method for detecting toxoplasma gondii infection in preparing a biomarker or a diagnostic reagent of a toxoplasma gondii infection-associated signaling pathway, wherein the signaling pathway includes but is not limited to one or more of a Jak-STAT signaling pathway, a chemokine signaling pathway and a Toll-like receptor signaling pathway.

In a seventh aspect of the present invention, there is provided a screening method for Toxoplasma gondii infection animal host brain tissue differential expression gene according to the first aspect of the present invention, a target site for detecting Toxoplasma gondii infection according to the second aspect of the present invention, a primer for detecting Toxoplasma gondii infection according to the third aspect of the present invention, a kit for detecting Toxoplasma gondii infection according to the fourth aspect of the present invention, or a detection method for detecting Toxoplasma gondii infection according to the fifth aspect of the present invention, for preparing a Toxoplasma gondii infection-associated disease biomarker, a diagnostic agent or a targeted drug.

The invention researches the action mechanism between differential expression genes of a host and the Toxoplasma gondii by comparing the brain tissues of the Toxoplasma gondii-infected masked palm civet and the healthy palm civet and screening the differential expression genes, and provides a target for searching biomarkers. The screening method of Toxoplasma gondii-infected masked civet brain tissue differential expression genes provided by the invention can quickly and effectively carry out transcriptomics research, and can indirectly confirm whether the host is infected with Toxoplasma gondii by detecting the differential expression genes of the host, thereby providing reference for researching the mechanism of wild animal host brain injury caused by Toxoplasma gondii infection and finding new biomarkers.

Drawings

FIG. 1 is a statistical chart of gene expression value distribution of genes differentially expressed in brain tissue of a Toxoplasma gondii-infected masked racoon dog according to an embodiment of the present invention, wherein the left side is a Toxoplasma gondii-infected group, and the right side is a control group;

FIG. 2 is a volcano diagram of genes differentially expressed in Toxoplasma gondii-infected masked racoon dog brain tissue according to an embodiment of the present invention;

FIG. 3 is a GO analysis diagram of genes differentially expressed in Toxoplasma gondii-infected masked racoon dog brain tissue according to an embodiment of the present invention;

FIG. 4 is a diagram of the pathway analysis of Toxoplasma gondii-infected raccoon brain tissue differential expression genes provided in the embodiments of the present invention;

FIG. 5 is a comparison graph of the expression levels of Toxoplasma gondii-infected masked paguma larvata brain tissue differential expression gene qRT-PCR and RNA-seq provided in the embodiment of the present invention;

FIG. 6 is a Western blot result chart of Toxoplasma infected masked racoon dog brain tissue differential expression genes provided in the embodiments of the present invention;

FIG. 7 is the comparison graph of the expression of gene qRT-PCR and RNA-seq differentially expressed in brain tissue of other animal hosts infected by Toxoplasma gondii provided by the embodiment of the invention.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.

Example 1: screening of relevant immune target gene of raccoon dog after brain infection with toxoplasma gondii

1 materials and methods

1.1 materials

A clean-grade masked palm civet of 3-8 months old is purchased from a special breeding base of Shaoguan Jiahe, Guangdong province; toxoplasma ME49 strain, was maintained by parasite laboratory of the veterinary college of agriculture university, south China.

1.2 sample preparation

Establishment of toxoplasma infection masked palm civet model

12 SPF-grade male masked palm civets of 4 months age were purchased from a special breeding base of Shaoguan Jiahe, Guangdong province, and 6 masked as experimental groups, and the other 6 were control groups. In the experimental group, 40 cysts of Toxoplasma gondii ME49 strain were inoculated into mice by intragastric administration, and diluted to 5 mL/mouse with physiological saline brain homogenate. The control group was gavaged with the same amount of physiological saline into the inside of the paguma larvata, and the daily state of the paguma larvata was observed. The experimental experiment takes the raccoon dog as the anesthesia at the 35 th day after infection. In order to reduce individual difference, 6 paguma civets which are most remarkably changed in appearance are selected for anesthesia, and brain tissues of every 3 paguma civets are biologically repeated for three times. Grouping method of control group is as above. Taking out brain tissue rapidly in sterile environment, quick freezing with liquid nitrogen, and storing at-80 deg.C.

1.3RNA extraction

Total RNA was extracted from a sample of nutria brain tissue by Trizol method. Putting a sample into a mortar precooled by liquid nitrogen, adding a proper amount of liquid nitrogen, quickly grinding, transferring into a 2mL centrifuge tube treated by DEPC, adding 1mL Trizol (Invitrogen, CA, USA), uniformly mixing by vortex oscillation, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 12000rpm/min for 10min, transferring the supernatant into a new 1.5mL centrifuge tube, adding 0.2mL chloroform, covering the tube cap, shaking for 15s, mixing, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 12000rpm/min for 10min, transferring the supernatant into a new 1.5mL centrifuge tube, adding 0.5mL isopropanol, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 12000rpm/min for 15min, removing supernatant, and washing with 75% ethanol prepared from RNase-free water; centrifuging at 4 deg.C and 12000rpm/min for 15min, and removing supernatant; naturally drying at room temperature, dissolving with RNase-free water, and storing at-80 deg.C. The Agilent 2100Bioanalyzer and RNA6000 Nano LabChip kit (Agilent, CA, USA) was used to determine the quality and purity of the total RNA extracted.

1.4RNA library construction and sequencing

After extraction of total RNA from the sample, eukaryotic mRNA was enriched with magnetic beads carrying oligo (dT). The extracted mRNA is cleaved into small fragments by divalent cations at high temperature, and the 1 st cDNA strand is synthesized using these short sequences as templates and using a six-base random primer, followed by addition of buffer, dNTPs, RNase H and DNA polymerase I (Invitrogen, CA, USA) to synthesize the 2 nd cDNA strand. And (3) purifying double-stranded products by using AMPure XP beads, repairing the cohesive end of DNA into a flat end by using T4 DNA polymerase and Klenow DNA polymerase activity, adding a base A to the 3' end and adding a connector, carrying out fragment selection by using AMPure XP beads, carrying out PCR amplification to obtain a final sequencing library, carrying out sequencing by using Illumina Hiseq2000/2500 after the library quality is qualified, and carrying out sequencing reading to obtain a double-end 2x100 bp.

1.5 raw data analysis and sequence Assembly

Sequencing to obtain original sequencing sequences (raw reads), wherein low-quality reads with a joint, ploy-N and sequencing primers are contained in the raw sequences, and sequencing data must be filtered to obtain clean reads in order to ensure the quality of information analysis. Meanwhile, sequence repeats of Q20, Q30, GC content, and clean reads were calculated. All downstream analyses were based on high quality clean reads. Among the various programs available, we used the Trinity program (http:// trinitylrnaseq. sourceforce. net /). And (3) splicing clean reads, and using the transcript sequence obtained by Trinity splicing as a reference sequence for subsequent analysis. The longest transcript in each gene was taken as the Unigene for subsequent analysis.

1.6 functional annotation of transcripts

In order to obtain comprehensive gene function information, clear reads are compared with 5 databases for gene function annotation, and if at least 1 clear read of a certain gene is the only comparison (unique match) with a reference gene, the reference gene is defined as an expression gene. Functional annotation of sequences was performed based on the following database: SWISS-PROT (S wissProt protein sequence database), NCBI non-redundant nucleic acid database NR (non-redundant protein data base), KEGG (Kyoto Encyclopedia of Genes and genomes), KOG (Karyotic organisours group up database) and databases widely used for protein families and domains Pfam. All searches were done at Blast.

1.7 Gene abundance assessment and differentially expressed Gene threshold setting

Gene expression levels the abundance of gene expression is measured by FPKM values (Reads Per base of exon model Per Million mapped Reads), and the effects of sequencing depth and gene length on Reads counts are currently the most commonly used methods for assessing gene expression levels. The FPKM value of each gene was calculated using MARS (MA-plot-based method with random sampling model) model in the DEGseq program. If the FPKM value is more than 1000, it is considered to be a highly expressed gene. Multiple of Change (log)₂ ^{RPKM-PRU/RPKM-Control}) Based on normalized gene expression levels. And (4) counting the differential expression condition of the genes, wherein the screened genes are significant differential expression genes. In this experiment, an FDR (false discovery rate) was set<0.001 and | log₂ ^{fold change}And | ≧ 1 "is used as a threshold for judging the differentially expressed gene.

1.8 GO and KEGG pathway enrichment of differentially expressed genes

GO (Gene ontology) is an internationalized gene functional classification system. GO functional significance enrichment analysis is performed by mapping all significance differential expression genes to each term of a GO database (http:// www.geneontology.org /), calculating the number of genes of each term, and then applying a hyper-geometric test to find GO terms significantly enriched in significance differential expression genes compared with the whole genome background. GO term was judged to be significantly enriched with a corrected P value <0.05 as a threshold. The P value calculation formula is as follows:

wherein N represents the number of GO annotated genes in the genome, N represents the number of differentially expressed genes in N, M represents the number of specific GO annotated genes in the genome, and M represents the number of differentially expressed genes in the specific GO annotated genes in M.

KEGG is the main public database for pathway analysis (http:// www.genome.jp/KEGG /), helping us to better understand the process of coordinating different genes in organisms and making their respective biological functions. The pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways, finds pathways significantly enriched in significantly differentially expressed genes compared to the whole genome background with the corrected P value <0.05 as a threshold, and the calculation formula is as above.

1.9 real-time fluorescent quantitative PCR analysis to verify mRNA expression level

From the RNA-seq, we verified the gene expression level using real-time fluorescent quantitative PCR (qRT-PCR) analysis. Total RNA was extracted from Toxoplasma gondii ME49 strain-infected paguma larvata brain tissue by RNAioso Plus (Takara, Dalian, China) and finally resuspended in RNase-free water. The concentration and purity of total RNA were measured using an ultramicro spectrophotometer (Thermo, Scientific Nanodrop 2000, Waltham, MA, USA). With SYBR PrimeScript^TMcDNA was synthesized using RT Master Mix (Perfect Real Time) kit (TaKaRa, Dalian, China). We designed specific qPCR primers using Premier 5.0 software (Premier Biosoft International, Palo Alto, Calif., USA) based on the reference sequence of the gene, and the gene-specific qPCR primers and the length of the product of interest are shown in Table 1. The qPCR reaction was performed in a Rotor-Gene Q (Qiagen) real-time system using SYBR Green (SYBR Premix Ex Tag TMII; TaKaRa Bio) staining. qPCR reaction (20 μ L): 10 μ L SYBR Premix Ex TaqII, ddH₂O6. mu.L, 1. mu.L each of the upstream and downstream primers (10. mu. mol/L), and 2. mu.L of the template (cDNA concentration was diluted uniformly to 40 ng/. mu.L). qPCR procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30sec, annealing at 60 ℃ for 1min, and 40 cycles in total. Each sample was replicated three times. To standardize gene expression, beta-actin is used as reference gene and control group is used as standardThe relative expression amount of each gene was 2^–ΔΔCtAnd (4) calculating.

1.10 Western blot to verify the protein expression level of part of genes

3 differential expression genes related to immune regulation are selected for protein expression level identification (CCL8, CCL23 and TLR4), and similarly, beta-actin is selected as an experimental reference gene. The pretreated protein sample is subjected to 10% SDS-PAGE (polyacrylamide gel electrophoresis) at a voltage of 80V for 30min, and then adjusted to 120V for electrophoresis for 60 min. The nylon membrane (Roche, Indianapolis, USA) is cut into appropriate size, and discharged in the order of sponge-3 layers filter paper-SDS-PAGE albumin glue-nylon membrane-3 layers filter paper-sponge, and the membrane is transferred at 150mA for 45 min. The nylon membrane is placed in 5% skimmed milk which is precooled at 4 ℃ and contains 0.1% Tween-20, the skimmed milk is sealed for 1h, specific antibodies are respectively added for incubation for 2h, the skim milk comprises a mouse monoclonal antibody CCL5(1:800, Abcam), a rabbit monoclonal antibody CASP8(1:1000, Abcam), a mouse monoclonal antibody TLR4(1:400, Abcam) and an internal reference beta-actin (1:5000, Abcam), TBST is used for washing the membrane, TBST solution is replaced every 10min, and the process is repeated for 3 times. Goat anti-mouse and goat anti-rabbit IgG-HRP (horse radish peroxidase) (1:2500, Tiangen Biotech (Beijing) Co. Ltd., China) was then added and incubated at 37 ℃ for 1h, and the membrane washing procedure was repeated. The results were observed using DAB staining solution (Tiangen Biotech (Beijing) Co. Ltd., China) and ECL-plus Western Blotting detection and analysis System (Tiannon, Shanghai, China).

2 results

2.1 RNA extraction results and Mass analysis

The detection result shows that the total RNA solution has high purity (both OD260/OD280 and OD260/OD230 are more than 2.0), the concentration is more than or equal to 800 ng/muL, the RNA Integrity Number (RIN) value is more than or equal to 7, and rRNA 28S/18S is more than or equal to 0.8, as shown in Table 2, the integrity of the extracted total RNA is better, and the requirement of subsequent experiments is met.

TABLE 2 RNA extraction quality results

2.2 RNA sequencing and data Pre-processing

In this experiment, RNA-Seq technique was used to perform transcriptome sequencing of paguma larvata brain tissue before and after Toxoplasma gondii infection. Total RNA extracted from both sets of samples was used to construct an RNA library and sequenced using Illumina Hiseq2000/2500 to yield raw reads with data volume of approximately 48.17G. After data filtering, clear reads of about 39G are obtained, and the sequencing data amount of each sample is above 6G. Approximately 97.92% of the data was valid and could be analyzed further. The sequencing data quality preprocessing results are shown in table 3.

TABLE 3 summary of sequencing data quality pretreatment results

Q20%: the probability of misidentification is 1%, i.e. the error rate is 1%, or the accuracy is 99%;

q30%: the probability of false recognition is 0.1%, i.e. the error rate is 0.1%, or the accuracy is 99.9%;

2.3 Gene expression level analysis

In transcriptome, we used FPKM (fragments Per Kilobase of exon model Per Million mapped fragments) to assess the abundance of gene expression in samples. Through the new transcript construction, there were 69907 transcripts in total, yielding 29128 unigenes. To more intuitively observe the value distribution of the two sets of data, we plot a box plot to show the two sets of data, as shown in fig. 1. As can be seen from the figure, the two sets of data did not differ significantly as a whole.

2.4 Gene differential expression analysis

We set the threshold for the difference gene to P<0.05，|log₂ ^(fold ^change)≧ 1, FDR-corrected q-value<0.05, the significance threshold for differences meeting the condition is labeled significan. The toxoplasma infection group was analyzed for significantly differentially expressed genes compared to the control group, and a total of 2808 genes were significantly different in expression level. There were 1948 differentially expressed genes down-regulated and 860 up-regulated. Partial Up-and Down-Regulation of differentially expressed genes are listed in tables 4 and 5, respectively. Meanwhile, we observed the distribution of significantly differentially expressed genes from all detected genes more intuitively by plotting a volcanic chart, as shown in fig. 2.

Table 4 partial upregulated expression of differentially expressed genes

TABLE 5 partial downregulation of expressed differentially expressed genes

2.5 bioinformatic analysis of differentially expressed genes

Significant differentially expressed genes were further studied, each differentially expressed gene was aligned to the GO database (criteria for significance screening: P < 0.05). The total number of genes involved in GO annotation in the data was 4483, where the genes annotated as significant differences for a particular GO total 2808 genes, mainly involved in stress response, immune response, cell proliferation and adhesion, apoptosis, etc. The total number of significant GO term genes with up-regulated difference genes is 860, and the total number of down-regulated difference genes is 1948.

Figure 3 is a map of GO term profiles with the most significant number of differentially expressed genes in the GO classification, where immune system processes, stress response and signaling are predominant in biological processes, cells, intracellular domains and organelles are predominant in cellular components, and protein binding, ion binding and signal transduction activities are involved in molecular function.

Genes generally have their biological functions, and KEGG pathway analysis can help us to better understand the biological functions of differentially expressed genes. Analysis of all the differential genes in the brain tissue of paguma larvata by the KEGG pathway (criterion for significance screening: P <0.05) resulted in a total of 4483 genes involved in KEGG annotation, where the total of 2808 genes annotated as significant differences for a particular KEGG. These differentially expressed genes were significantly enriched into 100 pathways. FIG. 4 is the most enriched pathway for a portion of differentially expressed genes, with the first 3 pathways annotated specifically to the KEGG with the most differentially expressed genes being the Jak-STAT signaling pathway (28), chemokine signaling pathway (23), and Toll-like receptor signaling pathway (19), respectively.

2.6qRT-PCR and Western blot validation

10 genes closely related to immunity are selected for qRT-PCR verification, and are compared with the result of RNA-seq, and the result is shown in figure 5. Since we did qRT-PCR, the expression levels of the genes were not completely consistent compared to RNA-seq, but the expression trends of these genes were consistent with the RNA-seq results. Further, Western blot was performed to select three differentially expressed genes (CCL8, CCL23, and TLR4) from the 8 genes to verify their protein expression levels, and the results are shown in fig. 6. Similarly, Western blot results were compared with RNA-seq results and we found that they were consistent in up/down regulation trend.

TABLE 6 primer design for qRT-PCR analysis

2.7 detection of the differentially expressed genes in the brain tissue of masked palm civet by qRT-PCR to determine whether Toxoplasma cells are infected with Toxoplasma cells

To further analyze the expression level changes of these genes in brain tissue after Toxoplasma gondii infection, we prepared BALB/c mouse brain tissue samples in the same way, and obtained brain tissue samples from bat, hedgehog and bamboo rat, smelly shrew, brown rat and black-edged tooth rat, which detected Toxoplasma gondii infection, extracted total RNA of the samples by Trizol method, and performed qRT-PCR verification using specific primers of each gene in Table 6. The reaction system, procedure and calculation method are the same as above. We used the primers of these genes to verify that Toxoplasma gondii ME49 strain infected the brain tissue of BALB/c mouse, and found bat, hedgehog and bamboo ratThe expression change trends of these genes in the test samples of smelly shrew, rattus norvegicus and odontobutis nigricans were also substantially identical to those of RNA-seq, and the results are shown in FIG. 7, in which the solid line indicates log₂ ^{(fold change)}Dotted line represents log ═ 1₂ ^{(fold change)}If the qPCR result of each test sample is greater than 1 or less than-1, it is considered to be significantly up-or down-regulated. In the infection group, 6 genes such as CCL5, CCL8, CCL23, CASP8, TLR1 and TLR4 are up-regulated in expression, STAT1, ROS1, NOS1 and NOS2 are down-regulated in expression, and experimental results prove that whether host brain tissues are infected by Toxoplasma gondii can be determined by detecting expression difference of 10 genes closely related to immune regulation in Table 6.

2.8 evaluation of detection aging by qPCR method for brain tissue Toxoplasma gondii infection related target gene

In order to further test the detection timeliness of the qPCR method provided by the present invention for target genes closely related to immune modulation, we divided 20 2 month-old masked palm civets into 11 groups of 2 animals each, and extracted cerebrospinal fluid for detection. The transcriptional levels of genes CCL5, CCL8, CCL23, CASP8, TLR1, TLR4, STAT1, ROS1, NOS1, and NOS2 were measured in 1d, 3d, 4d, 5d, 6d, 7d, 8d, 9d, 10d, and 12d of mouse brain tissue from toxoplasma ME 49-encapsulated (20) gavaged mice at the same time, as shown in table 7. A general PCR reaction is carried out by using a specific primer (Toxoplasma gondii GRA14 gene) in Chinese invention patent CN106834504A, a pair of specific primers (Toxoplasma gondii B1 gene) in Chinese invention patent CN107012237A and a qPCR of 10 target genes (closely related to immunity) which are differentially expressed by animal host brain tissues are used for carrying out a comparison experiment. As a result, when a blood sample is detected, the detection rate can be only 30% in 7 days by using a CN106834504A common PCR method, and can be only 30% in 6 days by using a CN107012237A fluorescent PCR method; in the examination of the cerebrospinal fluid and the brain tissue of the infected rat of the paguma larvata, the qPCR method of 10 target genes (closely related to immunity) differentially expressed by the animal host brain tissue provided by the invention can detect more than 60% of the target genes on the 5 th day of toxoplasma gondii infection, the detection rate on the sixth day is more than 80%, and 1-2 days ahead of the blood PCR or qPCR detection in CN106834504A and CN107012237A, the method has the advantage of detecting the toxoplasma gondii infection of the brain earlier, can judge the toxoplasma gondii infection condition of the host earlier and is more beneficial to the early prevention and control of toxoplasma gondii.

TABLE 7 comparison of blood and brain tissue samples from mice and masked palm civets infected with Toxoplasma gondii by four methods

Note: -represents negative; + represents positive; n represents that the used qPCR method is a specific molecular marker aiming at the immune system of the brain tissue of an animal host and cannot be applied to the detection of toxoplasma in blood; the numbers inside the brackets indicate "the number of animals with variation in gene differences/total number of animals".

Meanwhile, the qPCR method for detecting the target gene related to the brain tissue toxoplasma infection provided by the invention is used for detecting specificity verification, the mice are respectively gavaged with echinococcus granulosus, trichinella, cryptosporidium parvum, schistosoma japonicum, coccidium, brachypodium brachypomum, trypanosoma brucei and giardia lamblia, and the brain tissue of the mice is taken for detection after 12 days, and the specific result is shown in Table 8, which indicates that the method has extremely high detection specificity.

TABLE 8 differential Gene identification of differential expression in brain tissue following infection of mice with different parasites

Note: negative indicates that there was no differential expression change in the gene.

In conclusion, the method provided by the invention screens out the differentially expressed genes by comparing the brain tissues of the toxoplasma gondii-infected masked palm civet and the healthy masked palm civet, is beneficial to mining the immune regulation mechanism caused by the toxoplasma gondii-infected masked palm civet from the transcriptomics perspective, can provide a target for searching biomarkers, and can diagnose whether the host is infected with the toxoplasma gondii more early and accurately by detecting the differentially expressed genes of the host.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

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South China Agricultural University

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Claims

1. A screening method of Toxoplasma gondii infected animal host brain tissue differential expression gene, which comprises the following steps:

1) extracting animal host brain tissue RNA, constructing a library and sequencing transcriptome;

2) filtering sequencing original data, removing low-quality reads with a linker, ploy-N and sequencing primers, and calculating sequence repeatability of Q20, Q30, GC content and clean reads to obtain high-quality clean reads;

3) splicing clean reads by using a Trinity program to obtain a transcript sequence, and taking the longest transcript in each gene as a Unigene;

4) comparing clean reads to databases SWISS-PROT, NR, KEGG, KOG and Pfam for gene function annotation;

5) calculating the FPKM value of each gene by using a MARS model in a DEGseq program to evaluate the gene expression level, setting the FPKM value of more than 1000 as a high-expression gene, setting P to be less than 0.05, | log2(fold change) | > 1, setting the q value of FDR correction to be less than 0.05, marking the difference significant threshold meeting the conditions as significan, and screening the significant difference expression gene;

6) setting the standard of significance screening: p is less than 0.05, and gene functional classification is carried out on the significant difference expression genes through a GO database;

7) setting a standard of significance screening: p <0.05, pathway analysis was performed on significantly differentially expressed genes by KEGG database.

2. A target site for detecting toxoplasma infection, wherein the target site is a differentially expressed gene of brain tissue after toxoplasma infection of a host, comprising:

and/or

3. A primer for detecting toxoplasma infection, wherein the primer is designed for the target site of claim 2, comprising one or more of the following sets of primers 1) to 10):

1) a primer set for CCL5 as shown in SEQ ID NO. 1-2;

2) a primer set for CCL8 as shown in SEQ ID No. 3-4;

3) a primer set for CCL23 as set forth in SEQ ID nos. 5-6;

4) primer sets for CASP8 as set forth in SEQ ID nos. 7-8;

5) a primer set for TLR1 shown in SEQ ID NO. 9-10;

6) a primer set for TLR4 shown as SEQ ID NO. 11-12;

7) a primer set for STAT1 as set forth in SEQ ID nos. 13-14;

8) a primer set for ROS1 as shown in SEQ ID NO. 15-16;

9) primer sets for NOS1 as shown in SEQ ID NO. 17-18;

10) primer sets for NOS2 as shown in SEQ ID NO. 19-20.

4. A kit for detecting toxoplasma infection comprising the primer of claim 3.

5. A test method for detecting Toxoplasma gondii infection, which comprises using the gene for differential expression of the brain tissue of a Toxoplasma gondii-infected host according to claim 2 as a test subject;

when one or more genes in CCL5, CCL8, CCL23, CASP8, TLR1 and TLR4 are transcriptionally up-regulated and/or one or more genes in STA T1, ROS1, NOS1 and NOS2 are transcriptionally down-regulated, judging that the host brain is infected with the Toxoplasma gondii.

6. The method of claim 5, wherein the method uses the primer of claim 3 to detect host brain tissue.

7. The screening method of Toxoplasma gondii-infected animal host brain tissue differential expression gene according to claim 1, the target site for detecting Toxoplasma gondii infection according to claim 2, the primer for detecting Toxoplasma gondii infection according to claim 3, the kit for detecting Toxoplasma gondii infection according to claim 4 or the detection method for detecting Toxoplasma gondii infection according to claim 5, is used for preparing Toxoplasma gondii infection-associated signal pathway biomarker or diagnostic reagent, wherein the signal pathway includes but is not limited to Jak-STAT signal pathway, chemokine signal pathway, Toll-like receptor signal pathway.

8. The screening method of Toxoplasma gondii-infected animal host brain tissue differential expression gene according to claim 1, the target site for detecting Toxoplasma gondii infection according to claim 2, the primer for detecting Toxoplasma gondii infection according to claim 3, the kit for detecting Toxoplasma gondii infection according to claim 4 or the detection method for detecting Toxoplasma gondii infection according to claim 5, is used for preparing Toxoplasma gondii infection-associated disease biomarker, diagnostic reagent or targeted drug.