CN114686601B - A specific probe for detecting hemocyanin gene expression in Chinese mitten crab and its application - Google Patents

Abstract

A specific probe for detecting the expression of eriocheir sinensis hemocyanin genes and application thereof. Based on the principle of specific combination of probe sequence and mRNA of target gene in proportion, the fluorescent in situ hybridization technique is combined with image flow cytometry, different groups are distinguished by the presence or absence of fluorescent signals in cells, and whether different genes are expressed in the same cell can be known by expanding and utilizing multicolor fluorescence co-localization according to the functional difference of signal intensity analysis. Cell classification serves the needs of diagnosis and functional research. Conventional methods mostly employ antibody recognition to classify cells, but the current lack of commercially available antibodies by aquatic animals limits this application. The method does not need an antibody, starts from a gene sequence, prepares a fluorescent-labeled short probe, has mild hybridization conditions, can perform quantitative analysis on the level of an intact single cell, and has good application prospects in many aspects such as cell classification.

Description

A specific probe for detecting hemocyanin gene expression in Chinese mitten crab and 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 supported by the Tianjin Application Basic and Frontier Technology Research Program (19JCYBJC29700), and the Tianjin Key Laboratory of Aquatic Ecology and Breeding 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 specific combination of probe sequences and target gene target sequences in proportion, and combining fluorescence in situ hybridization technology and flow cytometry detection technology. Novel automated detection methods for expression measurement and cell classification. This method does not require the use of antibodies to classify mitten crab blood cells. It only needs to obtain partial sequence information of the target gene to synthesize the probe sequence. Shorter probes can improve diffusion and hybridization efficiency and significantly shorten hybridization time; through a relatively mild and unique After the hybridization operation, specific cell groups and functions are analyzed based on the presence and strength of the probe signal. The present invention provides a new technical solution for crustacean immunology research.

Background technique

The classification and isolation of cells are the premise and foundation of functional research. 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 studies. Currently commonly used medical hematology methods such as antibody classification are not applicable to crustaceans such as shrimps and crabs. The main reason is the lack of commercially available classification-specific antibodies. On the other hand, the purpose of classifying blood cells is to study their functions, which themselves rely on key molecules. As far as crustaceans are concerned, hemocyanin is the main protein component in hemolymph. Current research has found that it is synthesized by hepatopancreatic cells and blood cells and then secreted extracellularly to perform its functions. Some studies have also found that hemocyanin also has phenoloxidase-like activity during the evolution of animals. Therefore, hemocyanin, as an important functional molecule, is a current research hotspot. New and more accurate measurement methods are also helpful. To analyze its principle 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 molluscs. It is colorless when deoxygenated and turns blue when combined with oxygen. Its biological function is mainly related to oxygen transport in the body, but research in recent years has gradually confirmed that hemocyanin is a multifunctional protein. In addition to its oxygen transport function, it is also involved in the maintenance of osmotic pressure, energy storage and molt regulation. and other additional functions, and even has similar activity and antibacterial efficacy to phenoloxidase, so hemocyanin is considered an important immune molecule in arthropods and molluscs. Bioinformatics analysis suggests that hemocyanin may have evolved from phenol oxidase, so the two are very similar in physical and chemical properties. Decker et al. found that hemocyanin molecules in wolf spiders can be hydrolyzed by trypsin or chymotrypsin into monophenol oxidase and O-bisphenol oxidase, thus exhibiting phenol oxidase 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 broken into peptide fragments 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 uses modern immunofluorescence technology, fluid mechanics, lasers, applied electronics, computers and other technologies and principles to measure cell length, width, size, Surface antigen recognition markers can be used for rapid, quantitative, multi-parameter detection. Information such as DNA ploidy and cell cycle can also be analyzed through quantitative detection of intracellular nucleic acids. Cytokines and adhesion molecules can also be analyzed with the help of specific labeling and staining methods. etc. functions. 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 incubating it with the test sample. Therefore, the antigen-antibody complex with labeled fluorescence can be passed through 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. This technology also helps to quickly and accurately analyze the rejection of transplanted organs, diagnose clinical diseases, explore the pathogenesis of diseases, and provide correct guidance for prognosis. As the demand for flow measurement increases in many aspects, the technology is also developing rapidly and iteratively. The multi-dimensional panoramic flow cytometer that has emerged in recent years is a new type of flow cytometer with added image auxiliary functions. It not only retains a series of advantages of conventional multi-color fluorescence flow cytometry such as high throughput and high speed, but also Based on this, the design of the measurement module was optimized, the liquid flow pipeline was adjusted, and the measurement module and multi-channel image acquisition function were added, so that on the basis of ensuring high-precision and high-throughput measurement, the data of each cell in each detection channel can be acquired. The images include more parametric features and are suitable for precise comparison of any group or even a single cell at any location selected in the analysis. Hu Jinli et al. and Ren Xingchao et al. used this technology to study the classification of blood cells in crustaceans and molluscs. With the help of more unique measurement characteristics, the blood cells were divided into four fine groups without using antibodies, which provided a basis for the research on blood cells in crustaceans and molluscs. The most refined quantitative grouping standard to date.

The function of blood cells depends to a large extent on their specifically expressed key genes. For the detection of intracellular gene expression, antibodies can be used to analyze the protein level, or probes can be used to measure the mRNA transcription level. The former requires long-term research accumulation to screen out specific antibodies against the target gene, while the latter only requires obtaining partial sequences, designing specific probes and coupling them with fluorescent groups and other labels before they can be used for detection, especially for transcriptome analysis. Basically, the intracellular expression of differential genes is verified and detected. 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 complexity of operations to obtain specific antibodies, the probe detection method has better adaptability. Probes can be designed by knowing the partial sequence, and the cost of chemical synthesis is also lower than that of monoclonal antibody preparation; in addition, On the one hand, compared to antibodies, probes have smaller molecular weights and can quickly enter cells and bind to target sequences after a short period of incubation. The strategy of using probes can better solve the shortcomings of lack of antibodies faced by aquatic animal blood cell analysis, which are important for aquatic animal cell research and health monitoring.

Measurement results based on a large number of cells can more accurately reflect the actual condition of the body. Therefore, the in situ flow cytometry method (fluorescenceinsituhybridization-flowcytometry, FISH/FC) combines the high-throughput capability of flow cytometry with the specificity of probe detection. Or flow fluorescent in situ hybridization (Flow-Fish) can better solve the problem of lack of antibodies. In 1969, Pardue, Gall and John respectively tried to establish in situ hybridization technology. Radioactively labeled DNA is used to bind the target DNA sequence, and then the actual location of the target gene is anchored using autoradiography, and the expression of the target gene is detected at the intact level of the cell for the first time. Boumam and Langer first coupled a fluorescein label with a probe in 1981 to achieve non-radioactive in situ hybridization. Giovannoni applied in situ hybridization technology to bacterial research in 1988, targeting rRNA sequences for microscopic detection of bacteria. With the continuous development of safer fluorescence technology, Delong first used fluorescently labeled oligonucleotide probe sequences to detect microbial cells in 1989. Flow fluorescence in situ hybridization (flow-FISH) is also a non-radioactive molecular genetics experimental technology developed based on the principle of in situ hybridization, adding the advantage of high throughput. In 1998, Rufer analyzed the length of intracellular telomeres through flow fluorescence in situ hybridization, proving that this strategy is 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, there is a lack of commercial antibodies for blood cell classification, and research on cell function classification based on cell surface antigens is still in its infancy. The blood cell analysis method commonly used in the medical field, which is based on cell surface characteristics and relies on antibody recognition to divide cells into different functional groups, has not been widely used in aquatic animal research. For a long time, research on immune cells in 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 largely affected the development of related research. Generally speaking, 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 will take 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 using hemocyanin as the target functional molecule has been established. It can not only be used to screen specific groups of cells, but also quantitatively analyze the relationship between gene expression and biological functions. Therefore, it can better solve the problem of crustaceans. Key issues facing blood cell research.

Contents of the invention

The purpose of this invention is to address the contradiction between the current 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 that utilizes the characteristics of hemocyanin gene expression in blood cells of specific groups of crustaceans, designs sequence-specific probes, identifies functional groups from mRNA expression levels, and understands the health status of crustaceans based on quantitative measurement results, establishing a detection scheme Automated analysis method combining sequence-specific fluorescent probes with image flow cytometry measurements.

In order to achieve the above objects, the present invention discloses the following technical contents:

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

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

The present invention also discloses the application of the probe sequence in the expression detection of crustacean hemocyanin and the analysis and screening of cell groups; wherein the crustaceans refer to Litopenaeus vannamei and Procambarus clarkii. The detection interval has a variety of crustaceans with the ability to specifically bind to the probe. The experimental results show that effective detection results can be obtained by optimizing the detection conditions based on the sequence matching between the probe and the target region of the gene from different species.

According to the Chinese mitten crab hemocyanin gene sequence, the present invention designs a detection probe in its conserved region, and the end is labeled with a fluorescent group to accurately screen cell groups with hemocyanin, and through the hemocyanin expression level in single cells Learn about the health of crustaceans. The present invention proceeds according to the following main steps:

(1) Based on 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 an end-coupled fluorescent group.

(2) Use anticoagulant to collect blood cells, wash and fix them, and perform permeabilization treatment to facilitate the probe to enter the cell during the hybridization process and bind to the target mRNA sequence.

(3) Using a method that combines equal-interval cooling and stepwise addition of deionized formamide, the complex structure is opened at high temperature for a short period of time, and then deionized formamide is used to lower the hybridization temperature to protect the cell morphology.

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

The preferred method of the present invention is as follows

(1) Probe preparation

Referring to the full-length sequences of the two Chinese mitten crab hemocyanin subunits MT989440.1 and JF802122.1 published by Genbank, the preferred probe sequence designed in their conserved regions is 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', and FAM is used at the end of the probe. Fluorophore labeling.

(2) Preparing samples

Anticoagulant was used to collect hemolymph from the sinusoids of Chinese mitten crab, and Carnoy's solution suitable for protecting and fixing nucleic acids was used to fix the blood cells; 0.1% concentration of NP-40 was used for permeabilization, and RNAse inhibitors were added to protect RNA.

(3) Probe hybridization

Use 2xSSC as the basic hybridization buffer, suspend the fixed and permeabilized cell samples, and raise the temperature to 70 degrees in the hybridization oven to eliminate complex secondary structures; use a method of equal intervals of cooling and stepwise addition of deionized formamide. Add deionized formamide five times and gradually lower the hybridization temperature to 40 degrees to complete specific hybridization.

(4) Detection and analysis

Measure the sample on the machine and collect information such as side scatter intensity, side scatter area, probe fluorescence intensity, and images of each channel. Refer to the patent "A Method and Application for Rapid Analysis of Crustacean Hemolymphocyte Groups and Numbers" (ZL2017 1 0583739.3) Method analysis and discussion of relevant data.

The invention is described in more detail as follows:

(1) Probe preparation

According to the two complete hemocyanin subunit coding sequences of Chinese mitten crab published by GenBank, MT989440.1 and JF802122.1, the common conserved segment was selected to design a specific probe sequence of 5'-GAAGATGTTATCCATGTACTTGTGGAG -3', with a length of 27 bases, GC content 40.7%, Tm value calculated by Primer Premier 5.0 software is 60.8°C; this probe can be compared with GenBank: MT989440.1 sequence 1141-1167th and GenBank: JF802122.1 sequence 1254- Binding at position 1280; there is no non-specificity in the polymerase chain reaction using the probe sequence as a forward primer or a single primer, using the complementary sequence downstream of the probe sequence as a reverse primer, or single-primer amplification without a 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 at HPLC level.

(2) Blood cell preparation

a) Using a disposable sterile syringe, press the anticoagulant (338 mmol/L sodium chloride, 115 mmol/L glucose, 30 mmol/L trisodium citrate, 10 mmol/L disodium ethylenediaminetetraacetate, pH= 7.0) The volume ratio to hemolymph is 1:1, minimally invasive blood collection from the blood sinus at the base of the animal's swimming or walking legs; centrifuge at 500g for 5 minutes at 4 degrees to collect blood cells.

b) Blood cell fixation

Wash the collected blood cells once with pre-cooled PBS, resuspend in 1.0 ml of ice-cold PBS, and add 0.4 mL of pre-cooled Caronys solution (the ratio of methanol to glacial acetic acid is 3:1) under low-speed vortexing before use. Prepare), fix in ice bath for 10 minutes, centrifuge at 500g for 5 minutes, discard the supernatant.

c) Hemocyte permeabilization

Resuspend the cells in 2xSSC buffer containing 0.1% NP-40, add RNAse inhibitor according to the concentration in the user manual, and permeabilize for 5 minutes; centrifuge at 500g for 5 minutes and discard the supernatant.

(3) Probe hybridization

Resuspend the cells in 200 μL hybridization buffer (2x SSC), raise the temperature to 70°C in a hybridization oven to eliminate complex intra-chain structures, and mix with an equal volume of hybridization buffer containing 0.5-5 μg/mL probe; then use an equal volume A method that combines gradient cooling with equal amounts of deionized formamide. During the 15-minute hybridization process, the hybridization oven controls the sample to cool down from 70°C to 40°C five times, and the deionized formamide concentration increases from 0% to a final concentration of 25 %-30%; lower the temperature by 6°C each time and add 1/5 of the final concentration of deionized formamide.

(4) Measurement analysis

After hybridization, cells were washed with PBS and resuspended, filtered through a 70 μm cell sieve to remove adherent cells, and the ZL 2017 10583739.3 patented analysis method was used to divide the cell groups by making a scatter plot of side scatter intensity versus side scatter area, 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 groups suggested by the present invention and the aforementioned groups to judge the reliability of the results of the present invention; different Information such as changes in hemocyanin expression in samples or before and after experiments can also be used to monitor the health status of experimental animals.

Figure 1 is the analysis results from normal Chinese mitten crab blood cells. The range indicated by P1 in Figure A indicates the cells with positive probe signal, accounting for 9.4% of all blood lymphocytes, that is, only a small part of the blood lymphocytes express hemocyanin. protein genes. Since hemocyanin is likely to originate from phenol oxidase and is mainly distributed outside cells, most studies believe that it is mainly synthesized by hepatopancreatic cells. Studies on the expression distribution of hemocyanin genes in various crustaceans have shown that it is present in blood cells. The expression level is lower than that of the hepatopancreas. The measurement results of the detection method of the present invention also indicate that only about 10% of blood lymphocytes express hemocyanin, which on the one hand explains that blood cells are not the main expression site of hemocyanin, and on the other hand also indicates that there are cells expressing hemocyanin. Specific types of blood cells. Further analysis of the side scatter characteristics of the blood cell sample spots with positive signals from the probe is shown in Figure B. It is found that almost all of these cells come from the large granular cells represented by the R4 group, accounting for 96.5%, while the other groups of cells almost do not contain Hemocyanin. On the one hand, this result shows that the measurement results of the functional classification method established in the present invention and the existing methods can mutually confirm each other, and on the other hand, it provides more comprehensive functional information for the cell groups classified by the existing methods. Since hemocyanin mainly exists extracellularly, combined with the above analysis, it can be considered that large granular cells have secretory functions and are mainly involved in humoral immunity. Figure 1 Probe detection of hemocyanin gene expression in Chinese mitten crab; (A) Proportion of positive cells in normal whole blood of Chinese mitten crab; P1 is the positive interval of hemocyanin probe, accounting for 9.4%. (B) Analysis of cell groups with positive hemocyanin probe signal in Mitochondo sinensis; R1 agranular cells; R2 small granular cells; R3 medium granular cells; R4 large granular cells. Among them, the R4 group has the best correlation with the hemocyanin probe signal, accounting for 96.5%. 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 the drawings

Figure 1 shows the probe detection of hemocyanin gene expression of Chinese mitten crab; (A) the proportion of positive cells in the whole blood of normal Chinese mitten crab; P1 is the positive interval of hemocyanin probe, accounting for 9.4%; ( B) Analysis of cell groups with positive signal of hemocyanin probe in Eriocheir sinensis; R1 non-granular cells; R2 small granular cells; R3 medium granular cells; R4 large granular cells; among which the R4 group accounts for 96.5%;

Figure 2 shows the probe detection of hemocyanin gene expression in Eriocheir sinensis after infection with Aeromonas hydrophila; (A) P1 is the positive interval of the hemocyanin gene probe signal, accounting for 12.8%. (B) Analysis of cell groups with positive signal of the Chinese mitten crab hemocyanin gene probe; R1 non-granular cells; R2 small granular cells; R3 medium granular cells; R4 large granular cells; the proportions of R2, R3 and R4 groups respectively. are 5.1%, 11.8% and 83.1%;

Figure 3 shows the effect of ammonia nitrogen stress on hemocyanin gene expression in Chinese mitten crab; (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 individuals under ammonia nitrogen stress. P1 is a positive cell, accounting for 3.5%.

Detailed ways

The principles and applications of the present invention will be further described below with reference to the examples. The solutions of the embodiments described here do not limit the present invention. Those skilled in the art can make improvements and changes to them according to the spirit of the present invention. These improvements and All changes should be deemed to be within the scope of the invention, and the scope and essence of the invention are defined by the claims.

Specifically, the reagents and materials used in this invention: PBS, 20 x SSC mother solution, deionized formamide, and centrifuge tubes were all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; RNAse inhibitors and PCR reaction master mix It was purchased from Promega Company, and the fluorescent labeled probe was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; Chinese mitten crab used in the experiment was purchased from Tianjin Wangdingdi Aquatic Products Wholesale Market.

Example 1

Effects of injection infection with Aeromonas hydrophila on blood cells of Chinese mitten crab

Materials and Methods

Experimental Materials

Chinese mitten crab, weight 50±20g. Aeromonas hydrophila strains were isolated and preserved by our laboratory. Reagent consumables and probes were purchased from relevant 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 anticoagulant composition and concentration are 338 mmol/L sodium chloride, 115 mmol/L glucose, 30 mmol/L trisodium citrate, 10 mmol/L disodium ethylenediaminetetraacetate, pH=7.0, Carnoy's fixed The ratio of liquid components methanol to glacial acetic acid is 3:1, and should be prepared before use.

experimental method

Select individual Chinese mitten crabs with no injuries on the body surface and normal vitality, and divide them into a control group and an experimental group. Inject 50 μL PBS or Aeromonas hydrophila (approximately 1x10^7 cfu) respectively. After 24 hours, follow the instructions of the present invention. Method to collect and analyze blood cells.

Blood cell collection. details as follows:

a) Select the Chinese mitten crabs from the control group and the experimental group respectively, use a sterile syringe, and collect 500 μL of blood from the blood sinus at the base of the legs of the Chinese mitten crab with a 1:1 volume of anticoagulant and hemolymph; 4 degrees , centrifuge at 500 g for 5 min, and collect blood cells.

b) Wash and resuspend the blood cells in pre-cooled PBS, adjust the density to approximately 1x10^6 cell/mL; add 0.4 times the volume of pre-cooled PBS under low-speed vortexing, 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 user manual; centrifuge at 500g for 5 minutes, discard the supernatant.

(3) Probe hybridization

Resuspend the cells in 200 μL hybridization buffer (2x SSC), raise the temperature to 70°C in a hybridization oven for 5 minutes to eliminate complex intra-chain structures, and mix with an equal volume of hybridization buffer containing 0.5-5 μg/mL probe before use. A method that combines isogradient cooling with equal amounts of deionized formamide, cooling from 70°C to 40°C in 5 times, increasing the concentration of deionized formamide from 0% to a final concentration of 30%; each time the temperature is lowered by 6°C and supplemented with 1 /5 final concentration of deionized formamide.

(4) Measurement and analysis

After hybridization, wash the cells with PBS and resuspend them. Filter them through a 70 μm cell sieve and put them on the machine. Collect signals such as side scattering and fluorescence intensity. Refer to the ZL 2017 1 0583739.3 patent method and use the side scattering intensity-side scattering area as scatter points. Figure, cell groups are divided according to the aggregation of cell-like points; on the other hand, the association of blood cells that mainly express the hemocyanin gene with the aforementioned groups is determined based on the intensity of the fluorescence signal in the cells, and compared with the control group Aeromonas hydrophila Whether the hemocyanin gene-expressing blood cell populations change before and after infection. For the results of the control group, refer to the analysis of Figure 1 in the instruction manual, and for the results of the experimental group, refer to Figure 2 in this example. The comprehensive analysis is as follows:

As can be seen from Figure 1, R4 is the cell group that mainly expresses the hemocyanin gene. Several groups from R1 to R3 hardly express hemocyanin. Since hemocyanin mainly exists extracellularly, the R4 group is considered to have secretory function. Figure 2 shows the measurement results of this embodiment. The proportion of cells P1 with positive probe signal in A increased from 9.4% in the control group to 12.8%, which is a large increase. Further group analysis found that cells with positive signals changed from being concentrated in the R4 group in the control group to being distributed in all R2-R4 groups in the experimental group, with the proportion of the R4 group falling from 96.5% to 83.1%. larger. Compared with the R2 group, more positive signal cells come from the mesogranular cells of the R3 group. The above results suggest that the R3 group and the R4 group have a closer functional relationship. They may be cells with similar functions or different differentiation stages of the same type of cells. They all play an important role in responding to pathogen invasion and participate in Humoral immunity. Figure 2 Probe detection of hemocyanin gene expression in Eriocheir sinensis after infection with Aeromonas hydrophila; (A) P1 is the positive interval of the hemocyanin gene probe signal, accounting for 12.8%. (B) Analysis of cell groups with positive signal from the Chinese mitten crab hemocyanin gene probe; R1 agranular cells; R2 small granular cells; R3 medium granular cells; R4 large granular cells. Compared with normal individuals, the R4 group in the hemolymph of experimental individuals is still the main blood cell expressing hemocyanin, but its proportion of positive signal cells is significantly reduced to 83.1%. The R2 and R3 groups express the hemocyanin gene. The proportions increased 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 that perform similar functions under specific stimulation conditions.

Example 2

Rapid analysis of the effects of ammonia nitrogen stress on hemocytes of Chinese mitten crab

Materials and Methods

Experimental Materials

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

experimental method

Select individual Chinese mitten crabs with no injuries on the body surface and normal vitality, and place them in water with an NH4+ concentration of 5 mg/L. After 24 hours of acute stimulation, blood cells are collected and analyzed according to the method in the instructions of the present invention.

blood cell collection

a) Select the Chinese mitten crabs from the control group and the experimental group respectively, use a sterile syringe, and collect 500 μL of blood from the blood sinus at the base of the legs of the Chinese mitten crab with a 1:1 volume of anticoagulant and hemolymph; 4 degrees , centrifuge at 500 g for 5 min, and collect blood cells.

b) Wash and resuspend blood cells in pre-cooled PBS, with a density of approximately 1x10^6 cell/mL; add 0.4 times the volume of pre-cooled PBS under low-speed vortexing, 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 user manual; centrifuge at 500g for 5 minutes, discard the supernatant.

(3) Probe hybridization

Resuspend the cells in 200 μL hybridization buffer (2x SSC), heat to 70°C in a hybridization oven for 5 minutes to eliminate complex structures, and mix with an equal volume of solution containing 1 μg/mL probe and a final concentration of 30% deionized formamide. Mix the hybridization buffer and add an effective dose of RNAse inhibitor; cool down the hybridization oven to 40°C and incubate for 15 minutes.

(4) Measurement and analysis

After hybridization, wash and resuspend the cells with PBS, filter them through a 70 μm cell sieve, and then put them on the machine. Only the fluorescence intensity signal is collected, and the proportion of positive signal cells in blood cells between the experimental group and the control group is compared to quickly understand the health status of the animal.

Figure 3 A and B respectively show the probe detection results of the blood cells of the experimental animals in the control group and the experimental group. This embodiment 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. changes to understand the health status of experimental animals. The results showed that there was only a slight difference between the proportion of positive cells in the control group, 11.3%, and the 9.4% result of the normal individuals tested mentioned in the previous instructions; after 24 hours of acute stimulation with high-concentration ammonia nitrogen in the experimental group, only The blood cells of 3.5% still expressed hemocyanin. Compared with the control group, most of the initial cell signals expressing hemocyanin disappeared, indicating that the cell function was seriously affected and the body was in a very unhealthy state. The above results indicate that the hemocyanin gene is an important indicator molecule in the response of Chinese mitten crab to ammonia nitrogen stimulation. This method can be used for health monitoring of ammonia nitrogen and other environmental factor stress conditions.

By using the probe analysis strategy of the present invention, the changing characteristics of key gene expression of farmed animals can be quickly understood in just 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 complicated multi-step analysis, it can intuitively reflect the health status of the body and the response level of key immune molecules, thereby providing a basis for monitoring at any time and grasping breeding as early as possible. The health of animals is of great significance.

Figure 3 Effect of ammonia nitrogen stress on hemocyanin gene expression in Chinese mitten crab; (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 individuals under ammonia nitrogen stress. P1 is a positive cell, accounting for 3.5%.

The probe of the present invention is used to analyze the hemocyanin gene expression status of Chinese mitten crab blood cells before and after concentration ammonia nitrogen stress, indicating that ammonia nitrogen stimulation seriously affects the normal function of blood cells. This method does not require more analysis steps, and only needs one step to analyze the hemocyanin gene according to the test sample. The difference in key gene expression between the product and the control group can be used to understand the health status of the animal.

SEQUENCE LISTING

<110> Tianjin Normal University

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

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 27

<212> DNA

<213> Artificial sequence

<400> 1

gaagatgtta tccatgtact tgtggag 27

Claims (2)

1. A specific probe for detecting the expression of eriocheir sinensis hemocyanin genes, which is characterized in that: the probe sequence 5'-GAAGATGTTATCCATGTACTTGTGGAG-3', SEQ ID NO 1 is completely complementary with the 1141-1167 th bit of the eriocheir sinensis hemocyanin subunit coding sequence GenBank MT989440.1 and the 1254-1280 th bit of the other complete hemocyanin subunit coding sequence GenBank of eriocheir sinensis JF802122.1 respectively; the probe was fluorescently labeled with 5-terminal FAM and purified by HPLC.

2. Use of the probe of claim 1 in crustacean hemocyanin expression detection and cell population analysis and screening; wherein the crustacean is Eriocheir sinensis.