CN114686601A - A specific probe for detecting the expression of mitten crab hemocyanin gene and its application - Google Patents

Abstract

A specific probe and application for detecting the expression of mitten crab hemocyanin gene. Based on the principle that the probe sequence and the mRNA of the target gene are specifically combined in proportion, the fluorescence in situ hybridization technology is combined with the image flow cytometry, and different groups are distinguished by the presence or absence of fluorescent signals in the cells, and the functional differences are analyzed according to the signal strength. It can also be extended to use multicolor fluorescence colocalization to understand whether different genes are expressed in the same cell. Cell sorting serves needs such as diagnostics and functional studies. Conventional methods mostly use antibody recognition to classify cells, but the lack of commercially available antibodies in aquatic animals limits this application. This method does not require antibodies, and starts from the gene sequence to prepare fluorescently labeled short probes. The hybridization conditions are mild, and the quantitative analysis can be carried out at the level of complete single cells. It has good application prospects in many aspects such as cell classification.

Description

A specific probe for detecting the expression of mitten crab hemocyanin gene and its application

This patent application was completed by the School of Life Sciences of Tianjin Normal University/Tianjin Key Laboratory of Animal and Plant Resistance, and was awarded the Tianjin Application Basic and Frontier Technology Research Program (19JCYBJC29700) and the Tianjin Key Laboratory of Aquatic Ecology and Aquaculture Open Fund (TJAE2015005). .

technical field

The invention belongs to the field of detection technology, and relates to a single-cell-level high-throughput gene based on the proportional specific combination of a probe sequence and a target gene target sequence, combining fluorescence in situ hybridization technology and flow cytometry detection technology Novel automated assays for expression measurement and cell classification. This method does not need to use antibodies to classify mitten crab blood cells, and only needs to obtain partial sequence information of the target gene, and then the probe sequence can be synthesized. The shorter probe can improve the diffusion and hybridization efficiency, and significantly shorten the hybridization time; After the hybridization operation, specific cell groups and functions are analyzed according to the presence and strength of probe signals. The invention provides a new technical solution for the research of crustacean immunology.

Background technique

The classification and separation of cells is the premise and foundation of functional research, and blood cells are the core of the immune system. Therefore, related technologies such as classification and identification of blood cells are the basic support for various animal immunology research. Currently commonly used medical hematology methods such as antibody classification are not suitable for crustaceans such as shrimp and crabs, mainly due to the lack of commercially available antibodies for classification. On the other hand, the purpose of sorting blood cells is to study their function, which itself relies on key molecules. As far as crustaceans are concerned, hemocyanin is the main protein component in hemolymph. Current studies have found that hemocyanin is synthesized by hepatopancreatic cells and blood cells and then secreted to extracellular to perform its functions. Some studies have also found that hemocyanin also has phenoloxidase-like activity during the evolution of accompanying animals. Therefore, hemocyanin, as an important functional molecule, is a current research hotspot, and new and more accurate measurement methods are also helpful. To analyze its mechanism of action and the regulatory mechanism involved in immune response.

Hemocyanin is a copper-containing respiratory protein widely distributed in the hemolymph of arthropods and mollusks. It is colorless when deoxygenated and turns blue when combined with oxygen. Its biological function is mainly related to the oxygen transport in the body, but research in recent years has gradually confirmed that hemocyanin is a multifunctional protein. In addition to the oxygen transport function, it is also involved in the maintenance of osmotic pressure, energy storage and molting regulation. Hemocyanin is considered to be an important immune molecule in arthropods and mollusks. Bioinformatics analysis suggests that hemocyanin may be evolved from phenol oxidase, so the two are very similar in physical and chemical properties. Decker et al. found that hemocyanin molecules in tarantulas can be hydrolyzed into monophenoloxidase and O-bisphenoloxidase by trypsin or chymotrypsin, thereby exhibiting phenoloxidase activity. Salvato et al. demonstrated that a slightly acidic environment of pH 6 can induce mollusk-derived hemocyanin to exhibit O-bisphenoloxidase activity, and unhydrolyzed hemocyanin can also exhibit phenoloxidase activity. In addition to its phenoloxidase activity, hemocyanin can also be cleaved into peptides with antifungal and bacterial functions under specific conditions to resist the invasion of pathogenic microorganisms.

Flow cytometer is an important detection equipment that combines advanced technologies in many fields. It can measure the length, width, size, and size of cells by using modern immunofluorescence technology, fluid mechanics, lasers, applied electronics and computers and other technologies and principles. Rapid, quantitative, multi-parameter detection of surface antigen recognition markers, etc., and can also analyze DNA ploidy and cell cycle information through quantitative detection of intracellular nucleic acids, and can also analyze cytokines and adhesion molecules by specific markers and staining methods. etc. function. The binding of the antibody to the corresponding antigen is specific. After the antibody is fluorescently labeled, it can bind to the specific epitope of the antigen by co-incubating with the tested sample. Therefore, the antigen-antibody complex with labeled fluorescence can pass through the flow cytometry. Cytometer for quantitative detection. Based on the principle of specific binding of antigens and antibodies, multiple antibodies can also be used separately or simultaneously for cell typing and more refined subpopulation analysis. The technology also helps to quickly and accurately analyze the rejection of transplanted organs, as well as to diagnose clinical diseases, explore the pathogenesis of diseases, and provide correct guidance for prognosis. As the demand for streaming measurement grows in many ways, the technology is also developing rapidly iteratively. The multi-dimensional panoramic flow cytometer that has appeared in recent years is a new type of flow cytometer with added image assistance functions. It not only retains a series of advantages such as high throughput and high speed of conventional multicolor fluorescence flow cytometry, but also here On this basis, the design of the measurement module is optimized, the liquid flow pipeline is adjusted, and the measurement module and the multi-channel image acquisition function are added, so that on the basis of ensuring high-precision and high-throughput measurement, it is possible to obtain information including each cell in each detection channel. More parametric features, including images, are suitable for precise comparison of any group or even a single cell selected at any location in the analysis. Hu Jinli et al. and Ren Xingchao et al. applied this technique to the study of blood cell classification of crustaceans and mollusks. With the help of more unique measurement characteristics, blood cells were divided into 4 fine groups without using antibodies, which provided the research on blood cells of crustaceans and mollusks. The most refined quantitative grouping criteria to date.

The function of blood cells depends to a large extent on the key genes that are specifically expressed. For the detection of intracellular gene expression, antibodies can be used to analyze the protein level, and probes can be used to measure the mRNA transcription level. The former requires long-term research and accumulation to screen out specific antibodies against target genes, while the latter only needs to obtain partial sequences, design specific probes, and conjugate fluorescent groups and other labels for detection, especially for transcriptome analysis. Based on the verification and detection of the intracellular expression of the differential genes. The above analysis shows that both probes and antibodies can be used for gene expression analysis and complement each other. Compared with the screening cycle and the complexity of the operation to obtain specific antibodies, the probe detection method has better adaptability. The probe can be designed by knowing the partial sequence, and the cost of chemical synthesis is also lower than that of the preparation of monoclonal antibodies. On the one hand, the molecular weight of the probe is smaller than that of the antibody, and it can quickly enter the cell and bind to the target sequence after a short incubation time. Strategies using probes could better address the shortcoming of lack of antibodies for blood cell analysis in aquatic animals, which are important for aquatic animal cell research as well as for health monitoring.

Measurement results based on a large number of cells can more accurately reflect the actual situation of the body, so the in situ flow cytometry (fluorescenceinsituhybridization-flowcytometry, FISH/FC) method combines the high-throughput capability of flow cytometry with the specificity of probe detection. Or flow fluorescent in situ hybridization (flow fluorescent in situhybrid-ization, Flow-Fish) can better solve the problem of lack of antibodies. In 1969, Pardue and Gall and John tried to establish in situ hybridization technology respectively. Using radiolabeled DNA to bind the target DNA sequence, and then anchoring the actual position of the target gene by means of autoradiography, the expression of the target gene was detected at the cellular intact level for the first time. In 1981, Boumam and Langer first combined fluorescein labels with probes to achieve non-radioactive in situ hybridization. Giovannoni used in situ hybridization for bacterial research in 1988, targeting rRNA sequences for microscopic probing of bacteria. With the continuous development of safer fluorescent technology, in 1989, Delong used fluorescently labeled oligonucleotide probe sequences to detect microbial cells for the first time. Flow fluorescence in situ hybridization (flow-FISH) is also a non-radioactive molecular genetics experimental technology developed on the basis of the principle of in situ hybridization, which increases the advantages of high throughput. In 1998, Rufer analyzed the length of intracellular telomeres by flow fluorescence in situ hybridization, which proved that the strategy was sensitive, reliable, reproducible and easy to operate. Huang Xin also further confirmed that the detection results of the flow fluorescence in situ hybridization method are basically consistent with the measurement results of the real-time fluorescence quantitative PCR method.

Aquatic animal immunology research is relatively lagging behind, blood cell classification lacks commercial antibodies, and cell function classification research based on cell surface antigens is still in its infancy. Blood cell analysis methods commonly used in the medical field, which are based on cell surface characteristics and identify cells into different functional groups by means of antibody recognition, have not been widely used in aquatic animal research. For a long time, the research on immune cells of aquatic animals has mainly relied on manual methods such as staining and microscopy. The bottleneck of high-throughput automated classification and analysis technology has also greatly affected the development of related research. In general, aquatic animal immunology research urgently needs to solve the technical problem of functional classification first, and the development of a series of commercial antibodies for classification takes a long time. Therefore, the present invention takes into account both the needs of cell classification and functional analysis. A single-cell high-throughput analysis method with hemocyanin as the target functional molecule has been established, which can not only be used for the screening of specific groups of cells, but also quantitatively analyze the relationship between the gene expression and biological function, so it can better solve the problem of crustaceans. Key issues facing blood cell research.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the contradiction between the urgent need for automated high-throughput blood cell classification and measurement technology in the fields of crustacean immunology research and aquaculture health monitoring and the lack of suitable commercially available antibodies and the bottleneck of conventional methods. , proposed a detection scheme for using the characteristics of the hemocyanin gene expressed in blood cells of specific groups of crustaceans to design sequence-specific probes to identify functional groups from mRNA expression levels, and to understand the health status of crustaceans based on quantitative measurement results. An automated analytical method that combines sequence-specific fluorescent probes with image flow cytometry measurements.

To achieve the above object, the present invention discloses the following technical contents:

A specific probe for detecting the expression of Eriocheir sinensis hemocyanin gene, characterized in that: the probe sequence is 5'-GAAGATGTTATCCATGTACTTTGGGAG-3', SEQ ID NO: 1, and the probe sequence is respectively the same as that of Eriocheir sinensis. GenBank: 1141-1167 of the coding sequence of hemocyanin subunit of cheetah crab: 1141-1167 of MT989440.1 and another complete coding sequence of hemocyanin subunit of Chinese mitten crab GenBank: 1254-1280 of the base sequence of JF802122.1 Completely complementary; the probe is fluorescently labeled with 5-terminal FAM and purified by HPLC.

The invention further discloses a method for using the probe sequence, which is characterized by adopting a method of combining temperature reduction at equal intervals and stepwise addition of deionized formamide, that is, a hybridization process in which the temperature of the sample is controlled from 70 degrees Celsius to 40 degrees Celsius in a hybridization furnace The unique way of adding 20% final content of deionized formamide to the hybridization system each time is divided into 5 times at equal time intervals. Deionized formamide can reduce the characteristics of the hybridization temperature.

The invention also discloses the application of the probe sequence in the detection of crustacean hemocyanin expression and the analysis and screening of cell groups; wherein the crustacean refers to Litopenaeus vannamei and Crayfish. The detection section has a variety of crustaceans that have the ability to specifically bind to the probe. The experimental results show that the detection conditions can be optimized according to the sequence matching between the probe and the target region of the gene from different species, and effective detection results can be obtained.

According to the gene sequence of Chinese mitten crab hemocyanin, a detection probe is designed in its conserved region, and the end is labeled with a fluorescent group for accurate screening of cell groups with hemocyanin, and the expression level of hemocyanin in a single cell is determined by the detection probe. Learn about the health of crustaceans. The present invention is carried out according to the following main steps:

(1) According to the conserved region of the Chinese mitten crab hemocyanin subunit sequence published by Genbank, a probe was designed and synthesized, and the probe was labeled with a terminal coupled fluorescent group.

(2) Collect blood cells with anticoagulant, wash, fix, and then permeabilize, so that the probe can enter the cell during the hybridization process and bind to the target mRNA sequence.

(3) Using the method of cooling at equal intervals and adding deionized formamide step by step, the complex structure is opened at a high temperature in a short time, and then deionized formamide is used to reduce the hybridization temperature to protect the cell morphology.

(4) Parameter information such as side scatter and fluorescence intensity are measured on the cell machine for analysis.

The preferred method of the present invention is as follows

(1) Probe preparation

Referring to the full-length sequences MT989440.1 and JF802122.1 of the two Chinese mitten crab limpet hemocyanin subunits published by Genbank, the preferred probe sequence for the design of the conserved region is 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', and the end of the probe is FAM Fluorophore labeling.

(2) Preparation of samples

Anticoagulant was used to collect hemolymph from Sinus sinensis of Eriocheir sinensis, and blood cells were fixed with Carnot's solution suitable for protecting and fixing nucleic acid; NP-40 at a concentration of 0.1% was used for permeabilization, and RNAse inhibitor was added to protect RNA.

(3) Probe hybridization

Using 2xSSC as the base hybridization buffer, suspend the fixed and permeabilized cell samples, and heat up to 70 degrees in the hybridization oven to eliminate complex secondary structures; adopt the method of cooling at equal intervals and adding deionized formamide step by step, Deionized formamide was supplemented in 5 times and the hybridization temperature was gradually lowered to 40 degrees to complete the specific hybridization.

(4) Detection and analysis

Measure the sample on the computer, collect information such as side scatter intensity, side scatter area, probe fluorescence intensity, and images of each channel, refer to the patent (ZL2017 of "A method and application for rapid analysis of crustacean blood lymphocyte groups and numbers") 1 0583739.3) Method analysis to explore relevant data.

The present invention is described in more detail as follows:

(1) Probe preparation

According to the two complete hemocyanin subunit coding sequences MT989440.1 and JF802122.1 of Eriocheir sinensis published in GenBank, the common conserved segment was selected to design a specific probe sequence as 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', with a length of 5'-GAAGATGTTATCCATGTACTTGTGAG-3'. 27 bases, GC content is 40.7%, Tm value calculated by Primer Premier 5.0 software is 60.8℃; this probe can be respectively matched with GenBank: 1141-1167 of MT989440.1 sequence and GenBank: 1254-1254- of JF802122.1 sequence Binding at position 1280; no non-specificity in PCR amplification with the probe sequence as forward primer or single primer, with the downstream complementary sequence of the probe sequence as reverse primer, or single primer amplification without downstream primer The product was detected, indicating that the probe was specific; after synthesis, the probe was fluorescently labeled with 5-terminal FAM and purified by HPLC.

(2) Preparation of blood cells

a) Using a disposable sterile syringe, press anticoagulant (338 mmol/L sodium chloride, 115 mmol/L glucose, 30 mmol/L trisodium citrate, 10 mmol/L disodium EDTA, pH= 7.0) The volume ratio to hemolymph was 1:1, and blood was collected minimally from the blood sinus at the base of the swimming foot or walking foot of the animal; centrifuged at 4 degrees, 500 g for 5 min, and collected blood cells.

b) blood cell fixation

The collected blood cells were washed once with pre-cooled PBS, resuspended in 1.0 ml of ice-cold PBS, and 0.4 mL of pre-cooled Caronys solution (ratio of methanol to glacial acetic acid was 3:1, before use) was added under low-speed vortexing. Preparation), fixed in ice bath for 10 min, centrifuged at 500g for 5 min, and discarded the supernatant.

c) Hemocytopermeabilization

The cells were resuspended in 2xSSC buffer containing 0.1% NP-40, RNAse inhibitor was added according to the concentration in the manual, permeabilized for 5 minutes; centrifuged at 500g for 5 minutes, and the supernatant was discarded.

(3) Probe hybridization

Resuspend cells in 200 μL of hybridization buffer (2x SSC), warm to 70°C in a hybridization oven to eliminate complex intra-strand structures, and mix with an equal volume of hybridization buffer containing 0.5-5 μg/mL probe; The combined method of gradient cooling and the addition of equal amount of deionized formamide. During the 15-minute hybridization process, the hybridization furnace controlled the sample to cool down from 70°C to 40°C in 5 times, and the deionized formamide concentration was increased from 0% to the final concentration of 25°C. %-30%; each time the temperature was lowered by 6°C and supplemented with 1/5 final concentration of deionized formamide.

(4) Measurement analysis

After hybridization, the cells were washed with PBS and resuspended, and the adherent cells were removed by filtration through a 70 μm cell sieve. The ZL 2017 10583739.3 patented analysis method was used to make a scatter plot of the side scatter intensity against the side scatter area to divide the cell groups, and measure the fluorescence signal at the same time. The intensity in each cell; according to the signal strength of the hybridization probe, determine the blood cell group that mainly expresses hemocyanin, and analyze the correlation between the functional group suggested by the present invention and the aforementioned group, so as to judge the reliability of the result of the present invention; Information such as changes in the expression of hemocyanin before and after the sample or experiment can also be used to monitor the health status of experimental animals.

Figure 1 shows the analysis results of blood cells from normal mitten crab, in which the range indicated by P1 in Figure A represents the cells with positive probe signal, accounting for 9.4% of all blood lymphocytes, that is, only a small part of blood lymphocytes express blood blue protein gene. Since hemocyanin is likely to originate from phenol oxidase and is mainly distributed outside the cells, most studies believe that hemocyanin is mainly synthesized by hepatopancreatic cells. Studies on the expression and distribution of hemocyanin genes in various crustaceans have shown that it is highly expressed in blood cells. The expression level is lower than that of hepatopancreas. The measurement results of the detection method of the present invention also suggest that only about 10% of the blood lymphocytes express hemocyanin, which on the one hand explains that the blood cells are not the main expression site of hemocyanin, and on the other hand, it also indicates that there is a blood cell expressing hemocyanin. specific types of blood cells. Further analysis of the side scatter characteristics of the blood cell samples with positive signals of the probe is performed as shown in Figure B. It is found that these cells are almost all from the large granular cells represented by the R4 group, accounting for as high as 96.5%, while the other groups of cells hardly contain Hemocyanin. On the one hand, this result shows that the measurement results of the functional classification method established by the present invention can be mutually confirmed with the existing method, and on the other hand, it provides more comprehensive functional information for the cell groups classified by the existing method. Since hemocyanin mainly exists outside the cells, combined with the above analysis, it can be considered that the macrogranule cells have a secretory function and are mainly involved in humoral immunity. Figure 1. Probe detection of the expression of mitten crab hemocyanin gene; (A) the proportion of positive cells in the whole blood of normal mitten crab; P1 is the positive range of the hemocyanin probe, accounting for 9.4%. (B) Analysis of cell populations positive for mitten crab hemocyanin probe signal; R1 agranule cells; R2 small granulosa cells; R3 medium granulosa cells; R4 large granulosa cells. Among them, the R4 group has the best correlation with the hemocyanin probe signal, accounting for 96.5%, and it is a cell group that mainly expresses the hemocyanin gene. The R3 group accounted for only 2.4% of the positive signal cells, and no positive signal was detected in the other two groups.

Description of drawings

Figure 1 shows the probe detection of mitten crab hemocyanin gene expression; in which (A) the proportion of positive cells in normal mitten crab whole blood; P1 is the positive range of hemocyanin probe, accounting for 9.4%; ( B) Analysis of cell populations with positive signal from mitten crab hemocyanin probe; R1 agranulosa cells; R2 small granulosa cells; R3 medium granulosa cells; R4 large granulosa cells; the R4 population accounted for 96.5%;

Figure 2 shows the probe detection of mitten crab hemocyanin gene expression after Aeromonas hydrophila infection; (A) P1 is the positive range of hemocyanin gene probe signal, accounting for 12.8%. (B) Analysis of cell populations with positive signal from mitten crab hemocyanin gene probe; R1 agranule cells; R2 small granulosa cells; R3 medium granulosa cells; R4 large granulosa cells; the proportions of R2, R3 and R4 groups were respectively were 5.1%, 11.8% and 83.1%;

Figure 3 shows the effect of ammonia nitrogen stress on the expression of mitten crab hemocyanin gene; among them (A) probe detection of hemocyanin gene expression in normal individuals, P1 is a positive cell, accounting for 11.3%. (B) Probe detection of hemocyanin gene expression in ammonia-stressed individuals. P1 was positive cells, accounting for 3.5%.

Detailed ways

The principle and application of the present invention are further described below in conjunction with the examples. The solutions of the examples described herein do not limit the present invention. Those skilled in the art can improve and change it according to the spirit of the present invention. Variations should be considered to be within the scope of the invention, the scope and spirit of which is defined by the claims.

In particular, the reagents and materials used in the present invention: PBS, 20 x SSC stock solution, deionized formamide, and centrifuge tubes, etc. were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; RNAse inhibitor and PCR reaction premix It was purchased from Promega, and the fluorescently labeled probe was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; Eriocheir sinensis was purchased from Tianjin Wangdingdi Aquatic Products Wholesale Market.

Example 1

Effects of injection of Aeromonas hydrophila on blood cells of Eriocheir sinensis

Materials and Methods

Experimental Materials

Chinese mitten crab, weighing 50±20g. Aeromonas hydrophila was isolated and preserved by our laboratory. Reagent consumables and probes were purchased from related companies with reference to the instructions of the present invention. The probe sequence is: 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', which is fluorescently labeled with 5-terminal FAM and purified by HPLC. The composition and concentration of anticoagulant are 338 mmol/L sodium chloride, 115 mmol/L glucose, 30 mmol/L trisodium citrate, 10 mmol/L disodium EDTA, pH=7.0, Carnot's fix The ratio of methanol to glacial acetic acid is 3:1, which should be prepared before use.

experimental method

Chinese mitten crab individuals with no injury on the body surface and normal vitality were selected and divided into control group and experimental group, respectively injected with 50 μL of PBS or Aeromonas hydrophila (about 1×10^7 cfu), and 24 hours later, according to the instructions of the present invention, respectively. method to collect and analyze blood cells.

Blood cell collection. details as follows:

a) The control group and the experimental group were selected respectively, and 500 μL of blood was minimally collected from the blood sinus at the base of the foot of the Chinese mitten crab with a sterile syringe, with the volume of anticoagulant and hemolymph 1:1; 4 degrees , 500g centrifugation for 5min to collect blood cells.

b) Wash and resuspend blood cells with pre-cooled PBS, and adjust the density to about 1x10^6 cells/mL; add 0.4 times the volume of pre-cooled cells under low-speed vortex, fix in ice bath for 5 minutes, and centrifuge at 500g for 5 minutes.

c) Permeabilize cells with 0.1% NP-40 for 5 minutes, add RNAse inhibitor according to the concentration in the manual; centrifuge at 500 g for 5 minutes, discard the supernatant.

(3) Probe hybridization

Resuspend the cells with 200 μL of hybridization buffer (2x SSC), warm up to 70 °C in a hybridization oven for 5 min to eliminate complex intra-strand structures, mix with an equal volume of hybridization buffer containing 0.5-5 μg/mL probe, and then use The method of isocratic cooling combined with the addition of deionized formamide in equal amounts, cooling from 70 ℃ to 40 ℃ in 5 times, the deionized formamide concentration was increased from 0% to the final concentration of 30%; /5 final concentration of deionized formamide.

(4) Measurement and analysis

After hybridization, cells were washed with PBS and resuspended, filtered through a 70 μm cell sieve, and put on the machine to collect signals such as side scatter and fluorescence intensity. Refer to the patented method of ZL 2017 1 0583739.3, and use side scatter intensity-side scatter area as scatter points Figure, according to the clustering of cell-like points to divide the cell groups; on the other hand, according to the intensity of the fluorescent signal in the cells to determine the relationship between the blood cells that mainly express the hemocyanin gene and the aforementioned groups, and compare with the control group Aeromonas hydrophila Whether the blood cell populations expressing the hemocyanin gene change before and after infection. Refer to the analysis of FIG. 1 in the specification for the results of the control group, and refer to FIG. 2 in the present embodiment for the results of the experimental group. The comprehensive analysis is as follows:

It can be seen from Figure 1 that R4 is a cell group that mainly expresses the hemocyanin gene, and several groups of R1-R3 hardly express hemocyanin. Since hemocyanin mainly exists outside the cell, it is believed that the R4 group has a secretory function. Figure 2 presents the measurement results of this example. The proportion of cells P1 positive for the probe signal in A increased from 9.4% of the control group to 12.8%, a large increase. Further group analysis found that the cells with positive signals were concentrated in the R4 group from the control group to the R2-R4 group in the experimental group, and the proportion of the R4 group decreased from 96.5% to 83.1%, a decrease larger. Compared with the R2 group, more positive signal cells came from the R3 group mesogranule cells. The above results suggest that the R3 group and the R4 group are more closely related in function, which may be cells with similar functions or different differentiation stages of the same type of cells. They both play an important role in the process of responding to pathogen invasion and participate in the Humoral immunity. Fig. 2 Probe detection of hemocyanin gene expression in Eriocheir sinensis after Aeromonas hydrophila infection; (A) P1 is the positive range of hemocyanin gene probe signal, accounting for 12.8%. (B) Analysis of cell populations with positive signal from mitten crab hemocyanin gene probe; R1 agranule cells; R2 small granulosa cells; R3 medium granulosa cells; R4 large granulosa cells. Compared with normal individuals, the R4 group in the hemolymph of the experimental individual is still the blood cells that mainly express hemocyanin, but its proportion in the positive signal cells is greatly reduced to 83.1%, and the R2 and R3 groups express the cells of the hemocyanin gene. The proportion rose to 5.1% and 11.8% respectively. It is suggested that there is a certain functional correlation between the cell groups classified by flow cytometry, and they may not be completely independent cell types, and perform similar functions under specific stimulation conditions.

Example 2

Rapid Analysis of Effects of Ammonia Nitrogen Stress on Blood Cells of Eriocheir sinensis

Materials and Methods

Experimental Materials

Chinese mitten crab, weighing 50±20g. The ammonia nitrogen concentration is calculated according to the NH4+ concentration, and the stimulation concentration used in this example is 5 mg/L. Reagent consumables and fluorescent probes were purchased from related companies with reference to the instructions. The probe sequence was: 5'-GAAGATGTTATCCATGTACTTTGGGAG-3', which was labeled with 5-terminal FAM fluorescently and purified by HPLC. Anticoagulant component 338mmol/L sodium chloride, 115 mmol/L glucose, 30 mmol/L trisodium citrate, 10 mmol/L disodium EDTA, pH=7.0, Carnot's fixative component methanol The ratio to glacial acetic acid is 3:1, and it is prepared before use.

experimental method

The mitten crab individuals with no injury on the body surface and normal vitality were selected, placed in a water body with an NH4+ concentration of 5 mg/L, and 24 hours after acute stimulation, blood cells were collected and analyzed according to the method in the specification of the present invention.

blood cell collection

a) The control group and the experimental group were selected respectively, and 500 μL of blood was minimally collected from the blood sinus at the base of the foot of the Chinese mitten crab with a sterile syringe, with the volume of anticoagulant and hemolymph 1:1; 4 degrees , 500g centrifugation for 5min to collect blood cells.

b) Wash and resuspend blood cells with pre-cooled PBS at a density of about 1x10^6 cells/mL; add 0.4 times the volume of pre-cooled cells under low-speed vortex, fix in ice bath for 5 minutes, and centrifuge at 500g for 5 minutes.

c) Permeabilize cells with 0.1% NP-40 for 5 minutes, add RNAse inhibitor according to the concentration in the manual; centrifuge at 500 g for 5 minutes, discard the supernatant.

(3) Probe hybridization

Resuspend the cells with 200 μL hybridization buffer (2x SSC), warm up to 70 °C in a hybridization oven for 5 min to eliminate complex structures, and mix with an equal volume of 1 μg/mL probe and a final concentration of 30% deionized formamide. The hybridization buffer was mixed, and an effective dose of RNAse inhibitor was added; the hybridization oven was cooled to 40 °C and incubated for 15 minutes.

(4) Measurement and analysis

After hybridization, the cells were washed with PBS and resuspended, filtered through a 70 μm cell sieve, and put on the machine. Only the fluorescence intensity signal was collected, and the proportion of positive signal cells in the blood cells of the experimental group and the control group was compared, so as to quickly understand the health status of the animals.

A and B of Fig. 3 show the probe detection results of blood cells of experimental animals in the control group and the experimental group, respectively. This example adopts a rapid analysis strategy, that is, whether there is a significant difference in the proportion of cells expressing the hemocyanin gene in the whole blood only changes to understand the health of laboratory animals. The results showed that the proportion of positive cells in the control group was 11.3% and there was only a slight difference between the results of 9.4% of the normal individuals detected in the aforementioned instructions; after the experimental group was acutely stimulated with high concentrations of ammonia nitrogen for 24 hours, only The blood cells of 3.5 were still expressing hemocyanin. Compared with the control group, most of the cell signals that originally expressed hemocyanin disappeared, indicating that the cell function was severely affected and the body was in a very unhealthy state. The above results show that the limpet hemocyanin gene is an important indicator molecule in the process of the Chinese mitten crab responding to the stimulation of ammonia nitrogen, and this method can be used for the health monitoring of environmental factors such as ammonia nitrogen.

Using the probe analysis strategy of the present invention, the change characteristics of the key gene expression of the farmed animals can be quickly known in one step, and the health status of the animals can be evaluated accordingly. This example shows that the probe detection of the present invention is easy to operate, and the results have reference value. Even without complex multi-step analysis, it can intuitively reflect the health status of the body and the response level of key immune molecules, so as to monitor and grasp the breeding at any time as soon as possible. The health status of animals is of great significance.

Figure 3 The effect of ammonia nitrogen stress on the expression of hemocyanin gene in Eriocheir sinensis; (A) Probe detection of hemocyanin gene expression in normal individuals, P1 was positive cells, accounting for 11.3%. (B) Probe detection of hemocyanin gene expression in individuals under ammonia nitrogen stress, P1 was positive cells, accounting for 3.5%.

Using the probe of the present invention to analyze the expression status of hemocyanin gene in the blood cells of Eriocheir sinensis before and after the concentration of ammonia nitrogen stress, it shows that the ammonia nitrogen stimulation seriously affects the normal function of blood cells. The differences in the expression of key genes between the samples and the control group were used to understand the health status of the animals.

SEQUENCE LISTING

<110> Tianjin Normal University

<120> A specific probe for detecting the expression of mitten crab hemocyanin gene and its application

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 27

<212> DNA

<213> Artificial sequences

<400> 1

gaagatgtta tccatgtact tgtggag 27

Claims (3)

1. A specific probe for detecting eriocheir sinensis hemocyanin gene expression is characterized in that: the probe sequence 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', SEQ ID NO: 1 is completely complementary with the base sequence of the 1141-position 1167 of the eriocheir sinensis hemocyanin subunit coding sequence GenBank: MT989440.1 and the 1254-position 1280 of the JF802122.1 complete hemocyanin subunit coding sequence GenBank: JF802122.1 respectively; the probe is marked by 5-end FAM fluorescence and purified by HPLC grade.

2. A method for matching with the probe sequence of claim 1, which is characterized in that a method of combining equal interval temperature reduction with stepwise addition of deionized formamide is adopted, namely in the hybridization process of controlling the temperature of a sample to be reduced from 70 ℃ to 40 ℃ in a hybridization furnace, the deionized formamide can reduce the hybridization temperature by a unique mode of adding deionized formamide with the final content of 20% to a hybridization system every time in 5 times at equal time intervals.

3. The use of the probe sequence of claim 1 for the detection of hemocyanin expression in crustaceans and for the analysis and screening of cell populations; wherein the crustacean refers to Litopenaeus vannamei and Procambrus clarkii, and the crustaceans have specific binding ability with the probe of claim 1 in the detection region of the target gene.

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