CN114292871A

CN114292871A - Application of TLR4 gene in calf pneumonia resistant breeding

Info

Publication number: CN114292871A
Application number: CN202111534723.6A
Authority: CN
Inventors: 刘玲玲; 刘武军; 曹行; 马海玉; 王琼; 陈斌; 孙国智; 苏晓慧
Original assignee: Xinjiang Agricultural University
Current assignee: Xinjiang Agricultural University
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-04-08

Abstract

The invention provides application of a cattle TLR4 gene in calf pneumonia disease resistance breeding, the application verifies the condition of related protein expression after over-expression of TLR4 by co-transfecting a constructed over-expression vector pCMV-CYC-TLR4 into lung cells of a cattle, and detects the regulation and control effect on the related protein expression after over-expression of TLR4, compared with a control group, when the TLR4 gene is over-expressed, the expression activities of TIRAP, MYD88, NFKBIA, IRAK4, TRAF6, MAP3K7 and TAB2 genes are increased; the activity of TAB1 and IRAK1 genes is reduced. The activation and high expression of TLR4 are shown, and finally the high expression of an immune factor gene NFKBIA can be realized, so that a large amount of immune factors can be generated and participate in the immunity of an organism. The theoretical basis is provided for further research on the TLR4 on calf pneumonia disease resistance breeding at the organism level, and a reference is provided for breeding work for improving the calf disease resistance.

Description

Application of TLR4 gene in calf pneumonia resistant breeding

Technical Field

The invention relates to the technical field of animal genetic breeding, in particular to the technical field of cattle disease resistance breeding, and more particularly relates to application of a TLR4 gene in calf pneumonia resistance breeding.

Background

The calf pneumonia is an infectious disease in the feeding process of newborn calves, is a common disease, and is a main reason for causing the calf pneumonia due to small postnatal age, poor body resistance, incomplete development of organs and sensitivity to various stress factors of the calves. Commonly including lobar and lobular pneumonia and bronchopneumonia. Generally speaking, the incidence of the disease is highest in spring and winter, and the calf is seriously affected, so that secondary infection of digestive tract diseases or respiratory dysfunction is caused. The disease has high incidence, sudden onset and complex etiology. Pneumonia appears in calves in a breeding farm, other diseases are easily caused, especially secondary calf diseases, serious damage is caused to visceral organs, especially digestive tract organs and respiratory organs, and therefore the survival rate of the calves is seriously reduced. If the calf is not treated timely and effectively, the health of the calf is affected and huge economic losses are brought to farmers.

In the prior art, the prevention and treatment of calf pneumonia are mainly carried out by adopting a mode of sequential injection treatment of sodium chloride injection, gentamicin sulfate, 10% glucose injection, VC injection and composite VB injection, and a mode of combining Shuanghuanglian injection with other antibiotics or injecting etamsylate. Also adopts Maxingshigan powder, Yinqiao powder combined with radix Isatidis, fructus forsythiae, semen Armeniacae amarum, Scutellariae radix, radix Blueleaf, flos Lonicerae, Glycyrrhrizae radix, Gypsum Fibrosum, radix Platycodi, herba Ephedrae, radix Ophiopogonis, cortex Mori, etc. for treatment. Although the pneumonia onset symptoms of calves can be eliminated and recovered to a certain extent, the beef cattle fattening preparation has obvious influence on the fattening effect and the meat quality after recovery, and influences the sale of beef and the commercial value of the fattening cattle at the later stage.

Disclosure of Invention

The problems of the prior art cannot be solved radically for preventing and treating calf pneumonia, even after the calf pneumonia is cured, some adverse effects are still caused to the cattle raising industry, and disease-resistant breeding aiming at the calf pneumonia is not reported. Therefore, the technical problem to be solved by the invention is to overcome the defects in the prior art and provide the application of the bovine TLR4 gene in calf pneumonia disease-resistant breeding.

In order to solve the technical problems, the invention provides the following technical scheme:

the application provides application of a cattle TLR4 gene in calf pneumonia disease-resistant breeding.

Further, the application provides an application of the TLR4 gene of cattle in promoting the capability of lung cells of cattle in preventing bacterial infection.

Further, the present application provides an application of the above application in cattle breeding.

Furthermore, the application is that TLR4 further up-regulates TIRAP, MYD88, NFKBIA, IRAK4, TRAF6, MAP3K7 and TAB2 expression activity by inhibiting TAB1 and IRAK1 expression, thereby releasing a large amount of immune factors to prevent further infection of bacteria.

The application also provides a method for improving the capability of the bovine lung cells in preventing bacterial infection, which is to over-express the TLR4 gene in primary bovine lung cells.

Furthermore, the method for improving the capability of the bovine lung cells in preventing bacterial infection is to construct an overexpression vector pCMV-CYC-TLR4, transfect primary bovine lung cells and culture the cells.

The application also provides application of the bovine TLR4 gene as a target gene in preparation of a calf pneumonia treatment related preparation.

Furthermore, the bovine TLR4 gene is used as a target gene in preparation of calf pneumonia treatment related preparations, including but not limited to preparation of related vaccines, detection preparations and gene drugs.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1 is a graph showing the 48-hour growth of bovine lung cells.

FIG. 2 shows the structure of plasmid of pCMV-MYC-TLR4 overexpression vector.

FIG. 3 is a graph showing the relative quantification of bovine lung cell overexpression.

FIG. 4 is a graph showing the expression of the overexpression protein in bovine lung cells.

Figure 5 is a graph showing the proliferation and growth of TLR4 overexpression in bovine lung cells.

FIG. 6 is a graph showing the activity of TLR4 overexpression in bovine lung cells at various time points.

Figure 7 is a graph showing the effect of over-expressing TLR4 on the expression activity of the relevant genes.

Figure 8 is a graph showing the expression of different groups of TLR 4.

FIG. 9 is a graph comparing fluorescence quantification with transcriptome sequencing results.

Detailed Description

The present invention will be described in further detail below by way of examples. The design idea of the invention or simple substitution of the same belongs to the protection scope of the invention. The experimental procedures used below are, unless otherwise specified, all conventional procedures known in the art and the ingredients or materials used, if not specified, are all commercially available ingredients or materials.

The reagent and consumptive material that this application used are:

over-expression vector pCMV-MYC plasmid, Beijing Czejust corporation; DMEM high-glucose medium, DMEM-F12 medium, DPBS, Gibco company; pancreatin, Gibco; fetal bovine serum, Hangzhou Sijiqing Corp; double antibody, ampicillin, sigma corporation; MTT cell proliferation kit, CCK-8 kit, Biyuntian;

cell culture solution used in the present application: obtained by adding 50% fetal bovine serum and diabody (1/1000) to the basal medium and storing at 4 ℃.

Cell cryopreservation solution employed in this application: the culture medium is obtained by uniformly mixing a basic culture medium, DMSO and fetal calf serum according to the proportion of 7:2:1 and storing the mixture at the temperature of 4 ℃ in a dark place.

The instrument adopted in this application is: CO 2₂Incubator, SANYO corporation; clean bench, suzhou evolution equipment ltd; cell counting plate, Shanghai medical instrument factory; a constant temperature water bath DK98-1, tianjin instruments ltd; eppendorf centrifuge 5417R centrifuge, Thermo Electron Corporation, USA; tissue homogenizer, QIAGEN, germany; SIM-F124 Ice maker, SANYO, Japan; HVE-50 Autosterilizer, HIRAYAMA, Japan; MILLI-

Ultrapure water purification system, Millipor corporation, usa; alphalmager 2200 gel imaging System, Alpha Inc. USA; model DYY-6C electrophoresis apparatus, six instruments factories in Beijing; DYCP-31DN model electrophoresis tank, six instruments factories in Beijing; GL-861 vortex mixer, made by Jiangsu Lin Bell apparatus; an electronic balance: YP3001N, OMEGA; eppendorf pcr instrument, OMEGA; cell cryopreservation tubes, 0.22um filters, Gibco, Germany.

All reagents, instruments and materials selected for use in the present invention are well known in the art and are not intended to limit the practice of the present invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the present invention.

The first embodiment is as follows: application of bovine TLR4 gene in calf pneumonia resistant breeding

Example two: cell culture and passaging

1. Cell line culture and passaging

Bovine lung cells were from the national laboratory cell resource sharing platform. The basal medium of the bovine lung cells is a DMEM high-sugar medium, and the complete medium is prepared from 90% of the basal medium and 10% of fetal bovine serum.

2. Cell purification

Observing the climbing-out of the fiber cells, after the cells grow over a culture dish, carrying out digestion and passage on the cells by pancreatin for 2-3 times, removing suspended cells, and observing the cells after the cells are attached to the wall as lung cells according to the shape.

3. Cell passage

When the cell density reaches about 80%, cell passage can be carried out, and the specific passage steps are as follows: the old culture medium was aspirated and discarded, and carefully washed 3 times with PBS supplemented with double antibody; after digesting the cells for 3 minutes by adding 400. mu.L of 0.25% pancreatin containing EDTA and finding that the cells have changed from round to round, the edge of the culture dish is tapped to suspend the cells, 2 times the amount of the culture medium is added, and the dispersed cells are repeatedly blown and beaten. Half of the suspension was aspirated, inoculated into a new flask, observed under a microscope, and placed into a cell incubator for culture.

4. Cell freezing and recovery

Freezing and storing the cells: when the cell density reaches about 80%, the operation can be carried out, the cells are rinsed for 3 times by PBS, 0.25% of pancreatin containing EDTA is added for digestion, then the digestion is stopped by a whole culture medium, the cells are collected by centrifugation, then frozen stock solution containing 10% of DNSO (dimethyl sulfoxide) and 10% of fetal calf serum is added, the mixture is mixed uniformly and split packaged, the mixture is kept in a refrigerator at minus 80 ℃ overnight, and the mixture is put into liquid nitrogen for freezing and storing the next day.

And (3) recovering the cells: taking out the cell freezing tube from the liquid nitrogen, placing the tube into a 37 ℃ water bath kettle, shaking for 2 minutes, killing the freezing tube by using 75% alcohol, transferring the cell suspension into a 15mL centrifuge tube, adding 5mL culture medium, centrifuging at 800rpm for 5 minutes, and discarding the supernatant. Adding 5mL of culture medium, uniformly mixing, moving to a culture dish, putting into an incubator for culture, changing the culture medium for 24 hours, and observing the growth state of cells.

When the tissue block after the adherence is observed, after the culture for 24 hours or 48 hours in the incubator, cells around the tissue climb out and gradually extend, and growth halo appears. During the growth process, the liquid can be changed for half, and the cell passage can be carried out when the cell grows to fill the culture dish slowly. The cells have large shapes and clear outlines and are represented as slender flat structures. The cells can show slow growth in the first generation of passage, the full growth time of the cells is longer, the growth speed is accelerated after the cells enter a growth logarithmic phase, and the passage can be carried out in 48 hours. The observation of the state of the cells cultured for 48 hours is shown in appendix 1.

Example three: cell overexpression

1. Overexpression vector construction

An overexpression vector pCMV-MYC containing MYC label is designed into a gene TLR4 gene full-length CDS region, and an upstream primer and a downstream primer respectively contain EcoRI and KpnI enzyme cutting sites. The vector construction structure is shown in FIG. 2. PCR amplifies cDNA sequence, double enzyme digestion is carried out on pCMV-MYC plasmid, fragment size is determined, vector and fragment are recovered and then are connected by a Tiangen kit, and finally pCMV-MYC-TLR4 vector is constructed.

2. Transformation of

And transforming the constructed plasmid into an escherichia coli competent cell, culturing in an LB solid culture medium coated with ampicillin, and selecting a single colony for enzyme digestion and sequencing to identify a positive colony. Inoculating the positive bacteria into LB liquid culture medium containing ampicillin for expanding culture, shaking the bacteria in a shaker at 37 ℃ and 200rpm overnight, culturing for 12-14 hours, and extracting the plasmids by using the endotoxin-free plasmid extraction kit. And another bacterium liquid is taken for bacterium preservation, 1mL of shaken bacterium liquid is added into a 1.5mL EP tube, 250 mu L of glycerol is added, and the mixture is stored at the temperature of minus 20 ℃.

3. Transfection of overexpressing cells

Cells in good growth state were seeded in 24-well plates, and when the cells grew to 70-90%, the constructed overexpression vector was transfected into bovine lung cells according to liposome 3000(Lipofectamine 3000). According to the instruction, firstly using Opti-MEM culture medium to dilute and mix the Lipofectamine3000 reagent; then, the plasmid DNA was diluted with Opti-MEM medium to prepare a premix, the P3000TM reagent was added to the premix, the mixed plasmid DNA (1:1.5 ratio) was added to the diluted Lipofectamine3000 mixture, the mixture was incubated at room temperature for 10-15 minutes, the plasmid DNA-liposome mixture was carefully added to the cell culture dish to be transfected, and the culture was continued by changing the whole medium after 6 hours of incubation in the incubator. Transfected cells were harvested after 72 hours of culture.

The cells transfected with the overexpression vector were collected after 48 hours, and the overexpression vector was verified by a fluorescent quantitative PCR method, with transfection efficiency shown in FIG. 3. Collecting bovine lung cells transfected with the TLR4 gene overexpression vector for 48 hours, extracting RNA, performing reverse transcription, and detecting the expression condition of the TLR4 gene by using RT-PCR. After the cells are transfected with the over-expression vector, compared with the expression amount of the empty vector and the blank control group, the expression amount of the cells is obviously higher than that of the blank control group (P < 0.5).

Example four: western Blotting validation

1. Extraction of cellular proteins

Protein extract is extracted with mammalian extraction reagent (Thermo Fisher), and protease inhibitor (Supplemented with protease inhibitors, Roche) is added. The cells were first washed three times with PBS and then added with protein extract in an ice bath, then the cells were scraped off with a cell scraper, the mixture was recovered into a clean sterile 1.5mL centrifuge tube, incubated on ice for 20 minutes, then 13000rpm for 10 minutes, and the supernatant protein solution was removed to avoid contacting the bottom precipitate.

2、WB

Adding protein denaturant SDA-reducing 5X loading buffer into the extracted protein solution, then performing denaturation in a metal bath at 100 ℃ for 15 minutes, and immediately placing on ice after denaturation to prevent protein renaturation. The protein is transferred to a PVDF membrane after electrophoresis for 15 minutes at 120V and 55 minutes at 200V, and the membrane is transferred for 2 hours at constant current of 200mA in a wet mode and then is sealed for 2 hours. The primary antibody was incubated overnight at 4 ℃ and the membrane washed three times for 10 minutes each in TBST, followed by 1 hour incubation with secondary antibody and three membrane washes. And finally, mixing the developing solution 1:1 after preparation, uniformly dripping the mixture on a film, exposing the film by using a full-automatic chemiluminescence analyzer, and storing an image. The primary antibody was a Myc-tag antibody and the corresponding secondary antibody was HRP-labeled goat anti-mouse IgG.

And collecting transfected cells, extracting protein, performing denaturation treatment after protein concentration detection, and detecting the protein expression amount of the TLR4 gene in the over-expressed cells by using a WB method. As can be seen in bovine lung cells, the protein expression level of the overexpression vector was higher than that of the blank control group, and the results are shown in FIG. 4.

Example five: cell proliferation assay

Based on the first to fourth examples, the difference in the proliferation of cells in the case of overexpression was measured and detected by using an MTT cell proliferation assay kit. The cells with good growth conditions are inoculated into a 96-well plate, and the test can be carried out when the cell confluence degree reaches more than 70%. The over-expression vector pCMV-MYC-TLR4 vector plasmid and pCMV-MYC empty vector were manipulated according to the kit instructions. mu.L of MTT solution was added to each well of transfected cells and incubated in an incubator for 4 hours, at which time purple crystals appeared in the wells, followed by 100. mu.L of Formazan solution, shaking for 15 minutes on a shaker, and dissolving the crystals. And detecting the absorbance at 570nm by using a microplate reader, comparing the difference of the absorbance values of all groups, drawing a cell proliferation curve, and repeating the test for three times to eliminate errors.

And (3) measuring the absorbance of the cells by using a cell proliferation detection kit, detecting the proliferation condition of the cells in 4 time periods after the bovine lung cells are transfected, wherein the proliferation curve is shown as an attached figure 5, and the proliferation rate measurement condition is shown as a table 1. The number of the cells proliferated after the bovine lung cells are transfected with the overexpression vector is consistent.

Table 1: bovine lung cell TLR4 gene overexpression cell proliferation

	0h	24h	48h	72h
					pCMV-cyc	0.19±0.02	0.38±0.01	0.76±0.02	1.39±0.03*
TLR4	0.19±0.04	0.32±0.01	0.63±0.02	1.26±0.02

Note: indicates very significant correlation (P < 0.01); indicates significant correlation (P < 0.05). Fruit of Chinese wolfberry

Example six: cell viability assay

Based on the above examples one to five, the difference in viability of the cells in the case of overexpression was measured and detected by using CCK-8 detection kit. The cells with good growth conditions are inoculated into a 96-well plate, and the test can be carried out when the cell confluence degree reaches more than 70%. The over-expression vector pCMV-MYC-TLR4 vector plasmid and pCMV-MYC empty vector were manipulated according to the kit instructions. To the transfected cells, 10. mu.L of CCK-8 solution was added per well and cultured in an incubator for 4 hours, similarly to the method of cell proliferation assay. After the reagent is added, the absorbance at 450nm is detected by using a microplate reader at 0 hour, 24 hours, 48 hours and 72 hours respectively, the cell activity at each time point is calculated, the cell activity difference of each treatment group is compared, and the test is repeated three times to eliminate errors.

The activity of the transfected cells is detected according to a CCK-8 kit, OD values of four time points after transfection are measured by using a microplate reader, the measurement result is shown in figure 6, the transfection result in bovine lung cells can be obtained, and the overall trend of the cell activity is slightly lower than that of a control group when an overexpression vector is transfected for 72 hours.

Example seven: fluorescent quantitative PCR verification

Based on the first to sixth embodiments, the qPCR method is used for detecting the effect of the target Gene TLR4 in an NF-KB signal channel and the change of the expression level of TLR4 Gene with different expression levels on related downstream genes TAK1, TIRAP, TAB2, IRAK4 and NF-kappa B, and primers are designed by using AlleleID6.0 software according to the Gene sequence published by Gene Bank, and the primer sequences are shown in Table 2.

Table 2: primer sequence of target gene

The successfully constructed over-expression vector pCMV-CYC-TLR4 is transfected into bovine lung cells, and the influence of the TLR4 gene on downstream genes is detected by RT-PCR. The results are shown in figure 7, compared with the control group, when the TLR4 gene is over-expressed, the expression activity of TIRAP, MYD88, NFKBIA, IRAK4, TRAF6, MAP3K7 and TAB2 genes is increased; the activity of TAB1 and IRAK1 genes is reduced.

Example eight: application of bovine TLR4 gene in calf pneumonia resistant breeding

Three pneumonia attack calves and three healthy calves under the condition of consistent feeding management level are selected, wherein the samples of the sick group are marked as S1, S2 and S3, and the samples of the healthy group are marked as R1, R2 and R3. Blood samples are extracted by 5ml each, and the difference condition of TLR4 gene expression is detected by transcriptome sequencing, wherein the specific detection results are shown in Table 3:

table 3: transcriptome sequencing results

Sample name	TLR4
		S1	2992.221838
S2	3129.052993
		S3	3503.888133
R1	1450.335717
		R2	1890.072252
R3	1744.15343
		DS	3208.387655
DR	1694.8538
		log2FoldChange	0.920656593
pvalue	1.34E-06
		p_adj	0.000288964

From the above measurement results, it can be seen that the relative expression quantity multiple of the TLR4 gene between healthy and diseased calves is 1.8 times, and the difference in expression quantity between the two groups is extremely significant, as shown in fig. 8. Based on the qPCR method provided in the seventh embodiment, the qPCR detection method for detecting the target gene determines the expression of TLR4 between the two groups, and the result is compared with the sequencing result of the transcriptome, so that the result shows that the fluorescent quantitative PCR result and the sequencing result have the same trend of pneumonia susceptibility difference, and the transcription result is verified to be credible, as shown in FIG. 9. Therefore, relevant screening can be carried out in later breeding.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.

Claims

1. The application of the bovine TLR4 gene in calf pneumonia resistant breeding is provided.

2. Application of a bovine TLR4 gene in promoting bovine lung cells to prevent bacterial infection.

3. Use according to any one of claims 1 or 2 in cattle breeding.

4. The use as claimed in any one of claims 1 or 2 wherein TLR4 further upregulates TIRAP, MYD88, NFKBIA, IRAK4, TRAF6, MAP3K7, TAB2 expression activity by inhibiting TAB1 and IRAK1 expression, thereby releasing substantial amounts of immune factors to prevent further infection of the bacterium.

5. A method for improving the capability of bovine lung cells to prevent bacterial infection is characterized in that TLR4 gene is overexpressed in primary bovine lung cells.

6. The method of claim 5, wherein the cells are cultured by constructing the overexpression vector pCMV-CYC-TLR4 and transfecting primary bovine lung cells.

7. Use of bovine TLR4 gene as target gene in preparation of calf pneumonia treatment related preparation.

8. The use of the bovine TLR4 gene according to claim 7 as a target gene in the preparation of calf pneumonia treatment-related preparations, wherein the use includes but is not limited to the use in the preparation of related vaccines, detection preparations and gene drugs.