KR20060058249A - Specific expressed gene for fowl typhoid caused by invasion of salmonella gallinarum - Google Patents

Abstract

 Poultry typhoid, caused by Salmonella gallinalium bacteria, continues to spread throughout domestic poultry farms, and has been a national epidemic until recently. In particular, due to the large number of damages in laying hens, the economic loss in the domestic poultry industry is enormous. In recent years, the economic loss in the poultry industry is enormous. In recent years, the incidence and damage of young chicks from chicken breeding has increased rapidly. I'm doing it.

cDNA microarray technology compares the activity of thousands of genes continuously by arranging gene sequences of the same diameter on slide glass, and changes in environmental conditions such as changes caused by diseases such as poultry fever, E. coli, and vaccinia. It is widely used to observe changes in genes for. To date, research in the biological and molecular biological aspects of poultry has not been studied much compared to other fields. This idea is to observe biological and molecular biological changes using microarray technology for poultry typhoid chickens, a disease caused by Salmonella gallinalium infection in domestic chickens, and to specifically change genes and proteins that specifically change poultry. It is intended to be used as a diagnostic and indicator of typhoid fever.

Microarray, Poultry Fever, Salmonella Gallinium, Indicator, Chicken

Description

Specific expressed gene for fowl typhoid caused by invasion of Salmonella gallinarum}

 Poultry typhoid (FT) in poultry was first reported by Klein in 1889 as infectious enteritis from chickens, and similar diseases have been reported in brain birds and pheasants. Moore also described the disease as infectious leukemia, and in 1902 it was named 'fowl typhoid', which is used today by curtice, and soon afterwards the occurrence of poultry fever was reported in Germany and the Netherlands. The disease occurs worldwide, but in Canada. In countries such as the United States and many European countries, the incidence is very low or almost eradicated. It has been reported to increase rapidly in several countries in South America and Africa. Recently, new outbreaks of poultry fever have been confirmed in Europe, including Denmark and Germany.

In general, salmonella infections in poultry, including chicken, fall into three categories: pullorum disease (PD), FT, and paratyphoid. Among them, FT is Salmonella gallinaru m, which has the same antigenic structure as the standard type of salmonella pullorum , the causative agent of Chubaekri, and it is impossible to identify bacteria by morphological and immunological tests, but the resolution and biochemical properties of some sugars It is known that there is a difference between each other. In addition, there is a difference in disease susceptibility to infected hosts such as chickens. In PD, mainly chicks below 1 to 2 weeks of age are severely affected by the disease, and most of them are involuntary infections except in special cases. In the case of FT, it is known that the disease is susceptible to young chicks in the case of FT, and the susceptibility to disease increases as the age increases.

In the development of our country has never FT bars clearly revealed until the end of the 1980s, but just four weeks hayeotdago separated from the causative agent of the FT S.gallinarum chubaekri training based on the study of the anti-chubaekri separate from domestic reporting round in 1968 Investigations into the identification and development of these isolates have not been carried out. In 1992, the occurrence of this disease in Korea at that time was not reported at all, as there was no report on the occurrence of the disease and the isolation of causative organisms for more than 20 years until the domestic occurrence of FT was officially confirmed. It is estimated that there was no until.

The FT, which was confirmed in Korea in 1992, continues to spread to domestic poultry farmers, and it has been a national trend until recently. In particular, due to the large number of damages in laying hens, the economic loss in the domestic poultry industry is enormous. In recent years, the economic loss in the poultry industry is enormous. In recent years, the incidence and damage of young chicks from chicken breeding has increased rapidly. I'm doing it.                         

In order to eradicate the disease for a long time, various measures such as treatment and vaccination have been studied and reported by many people in many countries around the world, and it has been applied outdoors, but none of them have met the full expectations. Several poultry developed countries have established PD eradication programs at the national level for decades, and have carried out long-term killing policies for individuals or bacteria that are positive for Salmonella infection, reaching nearly the same level of disease cleansing.

In order to control domestic diseases of the liver, various preventive and therapeutic measures have been devised, including the development and dissemination of the bacteriophage vaccine, as well as the import and supply of bacillus and live vaccines from abroad, the selection of susceptible antibacterial agents, and the administration of high-level drugs. the causative agent of the infection during the in nature of the host Salmonella, including invasion S.gallinarum formed carriage shaft and a low immunogenicity, in particular the host cells to survive and proliferate, domestic Removing effective to control due to highly pathogenic in the spleen and liver It is not happening.

cDNA microarray technology compares the activity of thousands of genes in a row by arranging gene sequences of the same diameter on slide glass. This technique is widely used to observe changes in the genes caused by diseases such as poultry fever, E. coli, and bacteremia, and environmental changes such as stress. To date, research in the biological and molecular biological aspects of poultry has not been studied much compared to other fields. Therefore, in this study, microarray technology was used to observe biological and molecular biological changes in chickens infected with poultice, which is a disease caused by infection of Salmonella gallinarum in domestic chickens. It is intended to be used as a diagnosis and indicator of poultry fever.

Total RNA Isolation

Total RNA was extracted using Mini RNA Isolation IITM (Zymo Research, USA). 0.5 g of chicken liver was put in a tube and 600 µl of ZR RNA buffer was added and ground. After centrifugation at 2000 rpm for 5 minutes, only the supernatant was poured into the column and centrifuged at 13,000 rpm for 1 minute. 350 µl each of RNA Wash Buffer was added and centrifuged at 13,000 rpm for 1 minute. After repeating once, 50μl of RNase-free water was added and left for 5 minutes, followed by centrifugation at 13000rpm for 1 minute. The extracted RNA was measured for RNA concentration at 260nm by spectrophotometry. Total RNA was stored in -70 ℃ freezer until the experiment.

Chicken blood was heparin-treated and diluted with 10ml of PBS 20ml. 5 ml of Histopaque was added to a 15 ml centrifuge tube, and 10 ml of diluted blood was placed thereon to form a layer. After centrifugation at 2,500rpm for 30 minutes, the intermediate white blood cell layer was removed and placed in 50ml tube. Filled to 40ml with PBS and centrifuged at 1000rpm for 10 minutes, the supernatant was removed and suspended in pelllet 40ml of PBS. The supernatant was removed by centrifugation at 1000 rpm for 10 minutes, and 600 µl of ZR RNA buffer was added to the remaining pellet and ground. Subsequent procedures were identical to RNA isolation between chickens.

DNA chip fabrication

Gene expression was made by requesting Digital Genomes a spot containing 1120 chicken genes. The spotting of slides with cDNA was used Cartesian and the slides were corning GAPS II. The specifications of the fabricated chip were formatted in twin spot type with the same arrangement on one slide, and the diameter of each spot was adjusted to 130 ～ 160㎛. In addition, the distance between the four spots were set to 250 ㎛. The distance between blocks is 4.5mm and the distance of twin chip arrayrks is 21mm.

DNA chip

1) cDNA synthesis

 1μg of total RNA was added to the control group and Cyl primer was added to each control group. 2 μl of 5 × SuperScript II First Strand Buffer, 0.5 μl of dNTP mix, 0.5 μl of Superase-In RNAse Inhibitor and 0.5 μl of reverse transcriptase were added and reacted at 42 ° C. for 2 hours to synthesize cDNA. To stop the reaction, 1M NaOH / 100mM EDTA was added and reacted at 65 ° C. for 10 minutes to degrade RNA. After neutralizing 2M Tris-HCl (pH 7.5) was used for cDNA Hybridization.

2) cDNA Hybridization and Wash

 12.7 μl of synthesized control cDNA and 12.7 μl of experimental group cDNA were added to a new tube, and 27.4 μl of 2 × Enhanced cDNA Hybridization buffer and 2 μl of LNA dT Blocker were made, followed by reaction at 75 ° C. for 10 minutes. The cDNA Hybridization mix was placed on the Prewarmed Microarray and the coverslip was carefully covered to prevent bubbles from entering and then placed in a dark humidified chamber. The porcine chips used in the experiment were 70mer oligonucleotide spotted arrays and were reacted at 60 ° C for 16 hours.

After the reaction, the microarray was placed in a prewarmed 2 × SSC, 0.2% SDS wash buffer for 5 minutes, the coverslip was removed, washed for 20 minutes with 2 × SSC and 0.2% SDS wash buffer warmed to 42 ° C, and then washed with 20 × 2 × SSC wash buffer. Washed for a minute. Finally, 20 minutes were washed with 0.2 × SSC Wash buffer. Immediately after washing, centrifugation was performed at 900 rpm for 2 minutes to prevent staining of the microarray.

3) 3DNA Hybridization and Wash

 In the dark, 3DNA Array 900 Capture reagent Cy3 2.5 μl, Cy5 2.5 μl and 2 × SDS-Based Hybridization buffer 27.5 μl were mixed to create a 3DNA Hybridization mix. 0.275 μl of anti-fade reagent was added to reduce fading of fluorescent dyes post hybridization. After reacting the 3DNA Hybridization mix for 10 minutes at 75 ° C, it was placed on a Prewarmed Microarray and carefully covered with a coverslip. The microarray was placed in a dark humidified chamber and allowed to react at 60 ° C for 5 hours.

After the reaction, the microarray was placed in a prewarmed 2 × SSC, 0.2% SDS wash buffer for 5 minutes, and then the coverslip was removed. The microarray was washed with 2 × SSC and 0.2% SDS wash buffer warmed at 42 ° C. for 20 minutes, followed by 2 × SSC wash buffer. Wash for 20 minutes. Finally, 20 minutes were washed with 0.2 × SSC Wash buffer. Immediately centrifugation at 900 rpm for 2 minutes to prevent staining of the microarray. From 3DNA Hybridization, the experiment was performed in the dark room. To prevent the oxidation of fluorescent dyes attached to the microarray, DyeSaver2 was sprayed onto the microarray surface and coated.

4) Detection of emission signal

Using ScanArray 4000 (GSI Lumonics), Cy 3 was scanned at a wavelength of 550 nm and Cy5 was scanned at a wavelength of 650 nm to obtain fluorescent images. Results were analyzed using GenePix 5.0 software.

Analysis of cDNA microarray data

In order to normalize the expression patterns of each gene, this study used the global M method. First, normalization was performed by luminescence ratio between normal and poultry fever, and normalization was performed by comparing luminescence ratios with reference genes such as b-actin and GAPDH. Finally, the expression ratios in normal and poultry typhoid were converted to log / log ratios, and the log / log ratios of each gene were shown in scattered plots.

After normalizing the results on the DNA chip, spots were shown to show an increase in the emission ratio by more than 2 times and a decrease in the ratio below -0.5.                     

Analysis of Microarray

In order to observe changes in the liver of poultry typhoidosis caused by Salmonella gallinarum infection, cDNA microarrays containing cDNA and EST (expressed sequence tag) of chicken-related proteins were constructed. In order to observe protein changes in chicken liver, livers of chickens infected with normal and poultice were extracted and total RNA was extracted. After the hybridization between DNAs using the light emitters of Cy3 and Cy5 as markers, the emission ratio of the light emitters was measured using a detector. Fig. 1 shows the detection of Cy3 luminescent material on total RNA extracted from normal chicken liver, and the detection of Cy5 luminescent material on total RNA from chickens infected with poultry fever and detection of hybridization in microarray by detector. It was.

Spot detection with a microaed detector

The spots colored green are colored by the Cy3 emitter and are expressed in normal chickens.The spots colored red are colored by the Cy5 emitter and are expressed in poultry-infected chickens. have. Yellow spots are spots that show no difference in gene expression in normal chickens and chickens infected with poultry fever.

In order to normalize the expression patterns of each gene, this study used the global M method. First, normalization was performed by the luminescence ratio between normal and poultry fever, and normalization was performed by comparing the luminescence ratio with reference genes such as β-actin and GAPDH. Finally, the expression ratios in normal and poultry typhoid were converted to log / log ratios, and the log / log ratios of each gene are shown in the scattered plot in Figure 2.

After normalizing the results on the DNA chip, spots were shown to show an increase in the emission ratio by more than 2 times and a decrease in the ratio below -0.5 (Table 1). As a result, 41 genes showing significance were searched. Among them, three genes down-regulated by poultry typhoid were identified, and 38 genes were up-regulated.

The genes represented by the spots representing the results of the microarray analysis are shown in Table 1.                     

Comparing the expression of genes in normal chickens in poultry typhoid, it was found that the number of up-regulated genes was 3 and 38 compared to the dowun-regulated genes. Among the 41 genes, genes involved in immunity and metabolism in chickens were MEF2A protein, Norepinephrine transporter, Interleukin-16, DJ-1, Endothelin type A receptor, Interleukin-1 receptor I precursor and Mannose 6-Phosphate Receptor I could see that. The results suggest that the infection of poultry typhoid can lead to the deactivation of genes related to immunity and metabolism.

By using the difference in gene expression expressed by the poultry fever in the present invention can be used as an indicator for the infection and onset of the disease. In addition, it is possible to mass-produce the protein of the specifically expressed gene, and to prepare a strip-type diagnostic kit by preparing an antibody to the protein, it is possible to quickly diagnose poulticepus and infection.

Claims (4)

MEF2A gene specifically expressed by infection of poultry fever Protein synthesized from MEF2A gene Antibody made from protein of Clause 2 Strip-shaped diagnostic kit made using 3 antibodies