CN102732622A - Method of screening immunostimulation sensitive gene of Scylla paramamosain - Google Patents
Method of screening immunostimulation sensitive gene of Scylla paramamosain Download PDFInfo
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- CN102732622A CN102732622A CN2012101998656A CN201210199865A CN102732622A CN 102732622 A CN102732622 A CN 102732622A CN 2012101998656 A CN2012101998656 A CN 2012101998656A CN 201210199865 A CN201210199865 A CN 201210199865A CN 102732622 A CN102732622 A CN 102732622A
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Abstract
The invention relates to a method of screening an immunostimulation sensitive gene of Scylla paramamosain. The method comprises the following steps: (1) dividing Scylla paramamosain into an infection group and a control group, injecting the infection group with 100 mu L of Vibrio parahaemolyticus, injecting the control group with 100 mu L of normal saline, simultaneously treating the infection group and the control group for 24 h, respectively carrying out sampling at the time of 0 h, 1 h, 3 h, 6 h and 24 h, extracting the blood of Scylla paramamosain, extracting total RNAs of blood cells by using a Trizal method and preserving the total RNAs at a temperature of - 80 DEG C; and (2) designing multiplex gene expression primers with a candidate gene sequence used as a target sequence and the beta-actin gene used as an internal control gene, carrying out a reverse transcription reaction and a PCR reaction so as to obtain a PCR product, diluting the PCR product, loading the PCR product in a GeXP expression analysis system and determining the expression level of a candidate gene so as to obtain the immunostimulation sensitive gene. The method provided in the invention has the advantages of a simple process, low cost, capacity of rapidly screening the most sensitive gene from a number of related genes and a good application prospect.
Description
Technical field
The invention belongs to the sensitive gene of screening immunoreation field, particularly a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening.
Background technology
The immunity of crustacean lack of specific mainly relies on non-specific immunity to discern and foreign matters such as mikrobe of effectively removing invasion and parasite, and their immunity system mainly comprises cellular immunization and humoral immunization, and they are activated under immunostimulation.Cellular immunization mainly comprises formation and the package action etc. of engulfing, save knot.Humoral immunization mainly is to induce following some non-specific enzymes of synthesis secretion or the factor to wait the intrusion of resisting external germ in the stimulation of foreign matter.
Scylla paramamosain belongs to Arthropoda (Arthropoda), Crustachia (Crustacea), Decapoda (Decapoda), Portumidae (Portunidae); Mud crab belongs to (Scylla); Be the coastal important marine economy crab class of China, have bigger nutritive value and commodity value.Yet the frequent in recent years disease problem that takes place has seriously hindered the sound development of China's blue crab cultivation industry, in Zhejiang, lasting disease appearred in coastal main blue crab cultivation areas such as Fujian, Guangdong.Observe and predict data according to the hydrocoles disease, national mud crab average attack rate in 2006 is about 13.77%, and average mortality is 51.43%, and financial loss reaches 2.29 hundred million yuan, and the sound development of blue crab cultivation industry has been arrived in the outburst of disease serious threat.The mud crab disease of having reported has (Xu Haisheng etc., 2000 such as grasserie, brown spot, white awns disease, maculopathy, balantidiasis, vibriosis, parasitics dinoflagellate blood ovum whirlpool trichuriasis, aeromonas hydrophila disease; Wang Rongzhi etc., 2008).
In the face of severe diseases day by day in the blue crab cultivation process; The raiser is compelled against one's will and can only uses microbiotic; Yet antibiotic abuse has caused bacterium to produce very serious resistance; And also variety day by day of the cause of disease of cultivated crabs class disease outburst, this starts with from the immune defense of crab class self with regard to an urgent demand, improves crab class self disease resistance.The regulation and control and the expression of the disease resistance of crab class and himself immune correlation function gene have confidential relation.Therefore, screen and differentiate that immune correlation function gene is very important for the research of the natural disease resistance of crab class.
Multiple gene expression (GeXP) technology is a kind of brand-new gene expression profile quantitative analysis platform.The GeXP technology can detect 25 gene transcription levels of as many as in single RT-PCR reaction; It is the disruptive technology in multiplex PCR field; It combines multiple PCR technique and capillary electrophoresis technique cleverly; The method that adopts universal primer to combine with special primer, the consumption of its initial RNA can be low to moderate 10ng.The GeXP technology is the regeneration product of chip and Q-PCR technology, and it rises to a higher level to gene expression research; The number gene that this technology can be monitored can reach tens to hundreds of, and manageable sample number can reach thousands of.Based on the GeXP technology of RT-PCR provide a kind of sensitivity, gene quantification expression analysis technology flexibly and repeatably.It not only can handle a large amount of samples, and can handle simultaneously greater than 20 genes in a reaction, and can only handle 1 or several gene unlike the Q-PCR method at every turn.It is an ideal technique means that the multiple gene expression technology is screened the critical function gene.
Summary of the invention
Technical problem to be solved by this invention provides a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening; This method technology is simple; Cost is low, screens the most responsive gene of reaction in the heterogeneous correlation gene of can comforming rapidly, has a good application prospect.
A kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening of the present invention comprises:
(1) Scylla paramamosain is divided into infected group and control group, infected group is injected 100 μ L Vibrio parahaemolyticus, and control group is injected 100 μ L saline water, handles 24h simultaneously, respectively at 0h, and 1h, 3h, 6h, 24h sampling; Extract Scylla paramamosain blood, adopt the Trizal method to extract the total RNA of hemocyte, preserve down for-80 ℃;
(2) with the candidate gene sequence be target sequence, design multiple gene expression primer as internal control gene, carries out reverse transcription reaction and PCR reaction with beta-actin, obtains the PCR product; Appearance is gone up in PCR product dilution back, measure the expression level of candidate gene, promptly obtain the immunostimulation gene that is quick on the draw.
The concentration of Vibrio parahaemolyticus in the said step (1) is 2 * 10
6CFU/mL, the concentration of saline water is 0.9wt%.
The actual conditions of the reverse transcription reaction in the said step (2) is: 48 ℃ of 1min, 42 ℃ of 60min, 95 ℃ of 5min.
The actual conditions of the PCR reaction in the said step (2) is: 95 ℃ of 10min; 94 ℃ of 30s, 55 ℃ of 30s, 70 ℃ of 1min, 35 circulations.
The multiple gene expression primer sequence sees the following form:
The multiple gene expression primer that table 1 the present invention uses
Above-mentioned candidate gene sequence is seen sequence table SEQ ID No.19-26.
Beneficial effect
Technology of the present invention is simple; Cost is low; Can comform and screen the most responsive gene of immunoreation in the heterogeneous correlation gene rapidly; Set up the good technical platform for the immunologic function of further studying these genes, in the research field of Scylla paramamosain immunity system and its natural disease resistance, have a good application prospect.
Description of drawings
Fig. 1 is that 8 gene involved in immunity are expressed variation diagram behind the courses of infection; Wherein: proPO: pro-phenoloxidase; PPAF: pro-phenoloxidase incitant; α 2M: α macroglobulin; Lectin:C-type lectin; Trx 1: Trx; HSP70: HSP 70; HSP40: HSP 40; 70LHSP: type HSP 70 factors.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1, the experiment crab is prepared: with Scylla paramamosain adult (about body weight 300 grams) is subjects, totally 60, is divided into 2 groups (infected group and control groups), infected group injection Vibrio parahaemolyticus 100uL (2 * 10
6CFU/mL), control group injection 100uL saline water (concentration 0.9%) is handled 24h, respectively at 0h, and 1h, 3h, 6h, 24h sampling.
2, RNA extracts: extract mud crab blood, adopt the Trizal method to extract the total RNA of hemocyte ,-80 ℃ of preservations.
3, design of primers: pro-phenoloxidase, pro-phenoloxidase incitant, α macroglobulin, HSP 70, type HSP 70 factors, HSP 40, these 8 gene fragment orders of Trx; With these gene orders is target sequence, the multiple gene primer of operation eXpress Profiler software design on machine.
4, reverse transcription reaction: according to GenomeLab
TMThe operation of GeXP Start Kit specification sheets; Reaction system: DNase/RNase Free H2O 7 μ L, RT Buffer (5X) 4 μ L, Custom RT Rev Primer Plex 2 μ L, Reverse Transcriptase 1 μ L, KANrRNA with RI 1 μ L, Sample RNA 5 μ L.Reaction conditions: 48 ℃ of 1min, 42 ℃ of 60min, 95 ℃ of 5min.
5, PCR reaction: according to GenomeLab
TMThe operation of GeXP Start Kit specification sheets; Reaction system: PCR Buffer (5X) 4.0 μ L; 25mM MgCl2 (Thermo-Start) 4.0 μ L; Custom PCR Fwd Primer Plex 2.0 μ L; Thermo-Start DNA Polymerase (A25395) 0.7 μ L, cDNA Samples (RT reactions from the RT Plate) 9.3 μ L.Reaction conditions: 95 ℃ of 10min; 94 ℃ of 30s, 55 ℃ of 30s, 70 ℃ of 1min, 35 circulations.
6, the last appearance of GenomeLab: get the above-mentioned PCR product of 2 μ L and add 8 μ L water.Last appearance system is: dilution back PCR product 1.0 μ L, and DNA Size Standard-400 0.25 μ L, Sample Loading Solution 38.75 μ L, 1 in MO adds above-mentioned sample solution in the sample plane.With sample plane corresponding buffered liquid plate hole in add the damping fluid of 3/4 volume; Get into Set Up program, input sample name is specified the Frag-3 separation method to each sample, the GeXP analytical procedure of specify default, and program brings into operation.
7, data analysis: in excel, analyze the experimental data that obtains.Draw histogram, as shown in Figure 1, C-type lectin reached the highest at 1 hour, was far longer than other gene expression doses, and other genes just peaked at 3 hours or 6 hours, explained that this gene is to compare the sensitive gene at the courses of infection afterreaction.
Claims (7)
1. one kind is screened the be quick on the draw method of gene of Scylla paramamosain immunostimulation, comprising:
(1) Scylla paramamosain is divided into infected group and control group, infected group is injected 100 μ L Vibrio parahaemolyticus, and control group is injected 100 μ L saline water, handles 24h simultaneously, respectively at 0h, and 1h, 3h, 6h, 24h sampling; Extract Scylla paramamosain blood, adopt the Trizal method to extract the total RNA of hemocyte, preserve down for-80 ℃;
(2) with the candidate gene sequence be target sequence, beta-actin is as internal control gene, and design multiple gene expression primer carries out reverse transcription reaction and PCR reaction, obtains the PCR product; With PCR product dilution, in GeXP expression analysis system, go up appearance, measure the expression level of candidate gene, promptly obtain the immunostimulation gene that is quick on the draw.
2. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1, it is characterized in that: the concentration of the Vibrio parahaemolyticus in the said step (1) is 2 * 10
6CFU/mL, the concentration of saline water is 0.9wt%.
3. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1, it is characterized in that: candidate gene and multiple gene expression primer thereof in the said step (2) are respectively:
The α macroglobulin,
Forward primer: 5 '-AGGTGACACTATAGAATACCGGATAAGGATGACCTCAA-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA GGTGTCCTTCACCGTCAACT-3 ';
C-type lectin,
Forward primer: 5 '-AGGTGACACTATAGAATAAACTTTGTGATAATCGCCCG-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA CGACCTTCCACTTGTCCTGT-3 ';
HSP 70,
Forward primer: 5 '-AGGTGACACTATAGAATAAAGGACAAGGTGAGCGAAGA-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA TGCTCCAGTTCCTTCTGCTT-3 ';
HSP 40,
Forward primer: 5 '-AGGTGACACTATAGAATAGAGGAAGGGTGACATCATCG-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA TACCCACACTCCCTTTCTGG-3 ';
Class HSP 70 factors,
Forward primer: 5 '-AGGTGACACTATAGAATAATGCGAATGGTATCCTCCAG-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA TCCTGGACTCTACACGCTCC-3 ';
Trx,
Forward primer: 5 '-AGGTGACACTATAGAATAGTGGACGTGGATGAGAGTGA-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA TGATCCTGAGGGAAACCAAG-3 ';
The pro-phenoloxidase incitant,
Forward primer: 5 '-AGGTGACACTATAGAATATATCCTCAGACCTTCGCGTC-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA TTGTTGTCCAGGCAGATGAC-3 ';
Pro-phenoloxidase,
Forward primer: 5 '-AGGTGACACTATAGAATAAGGTAGCACAGCCAGATGGT-3 ',
Reverse primer: 5 '-GTACGACTCACTATAGGGA CATTGGTGAAGGTGATGGTG-3 '.
4. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1 is characterized in that: the forward primer of the beta-actin in the said step (2):
5’-AGGTGACACTATAGAATAGGTCGTACCACCGGTATTGT-3’；
Reverse primer: 5 '-GTACGACTCACTATAGGGA CTGGCCATCAGGAAGCTC-3 '.
5. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1, it is characterized in that: the actual conditions of the reverse transcription reaction in the said step (2) is: 48 ℃ of 1min, 42 ℃ of 60min, 95 ℃ of 5min.
6. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1, it is characterized in that: the actual conditions of the PCR reaction in the said step (2) is: 95 ℃ of 10min; 94 ℃ of 30s, 55 ℃ of 30s, 70 ℃ of 1min, 35 circulations.
7. a kind of be quick on the draw method of gene of Scylla paramamosain immunostimulation of screening according to claim 1; It is characterized in that: the appearance operating procedure of going up in the said step (2) is: PCR product after will dilute and dna molecular amount Marker mixture adding sample plane, with sample plane corresponding buffered liquid plate hole in the damping fluid of adding 3/4 volume; Get into Set Up program, input sample name is specified the Frag-3 separation method to each sample, the GeXP analytical procedure of specify default, and program brings into operation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317813A (en) * | 2019-07-17 | 2019-10-11 | 中国科学院海洋研究所 | Portunus trituberculatus Miers c-type agglutinin PtCLec2 gene and its coding albumen and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317813A (en) * | 2019-07-17 | 2019-10-11 | 中国科学院海洋研究所 | Portunus trituberculatus Miers c-type agglutinin PtCLec2 gene and its coding albumen and application |
| CN110317813B (en) * | 2019-07-17 | 2022-12-27 | 中国科学院海洋研究所 | Portunus trituberculatus C-type lectin PtCLec2 gene, and coding protein and application thereof |
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Application publication date: 20121017 |