CN103498003B - A kind of fluorescence RT-PCR detects the method for hybridized prussian carp TLR9 gene relative expression quantity - Google Patents

A kind of fluorescence RT-PCR detects the method for hybridized prussian carp TLR9 gene relative expression quantity Download PDF

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CN103498003B
CN103498003B CN201310502471.8A CN201310502471A CN103498003B CN 103498003 B CN103498003 B CN 103498003B CN 201310502471 A CN201310502471 A CN 201310502471A CN 103498003 B CN103498003 B CN 103498003B
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范玉顶
曾令兵
周勇
董圣
徐进
张辉
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Yangtze River Fisheries Research Institute CAFS
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Abstract

The invention discloses a kind of method that fluorescence RT-PCR detects hybridized prussian carp TLR9 gene relative expression quantity.By hybridized prussian carp TLR9 gene specific upstream and downstream primer: TLR9-qF:5 ’ – TCCAGAGTCATTGGCTGGTGTT – 3 '; TLR9-qR:5 ’ – CATAGAGATGCTTCAGGAGGGG – 3 ', obtains the 140bp product of hybridized prussian carp TLR9 gene.The gene expression abundance of TLR9 gene reflects the immune activity of hybridized prussian carp to a certain extent, can as the immunologic surveillance index in hybridized prussian carp diseases prevention and treatment.By detecting the change of hybridized prussian carp TLR9 gene expression amount, can judge whether hybridized prussian carp has infected cause of disease ahead of time, taking effective prophylactic treatment measure in time, avoiding the severe development of disease to cause irremediable massive losses.

Description

A kind of fluorescence RT-PCR detects the method for hybridized prussian carp TLR9 gene relative expression quantity
Technical field
The invention belongs to biological technical field, relating to the Auele Specific Primer for detecting hybridized prussian carp TLR9 genetic expression, more particularly relate to a kind of method that fluorescence RT-PCR detects hybridized prussian carp TLR9 gene relative expression quantity.Be mainly used in hybridized prussian carp TLR9 gene expression regulation study mechanism and immunologic function research, and the monitoring of the early stage pathogen infection of hybridized prussian carp.
Background technology
Along with the fast development of World Aquaculture, aquaculture production significantly increases, and the across national boundaries of fishery products and the trans-regional interchange of aquatic product sprout are also day by day frequent, greatly add the propagation probability of aquatic animal pathogenic micro-organism.In addition, the high-density of aquaculture is intensive, the abuse of the deterioration of Fishery Water Environment and various antibacterial agents class fishing medicine, also can cause the stress reaction of aquatic animal, cause the immunity system of body to be suppressed.The influencing each other of these factors just, causes fish disease to take place frequently, causes immeasurable loss to Fisheries Development.Use modern biotechnology as the technical support of aquaculture, the immune defense technology grasping disease is the problem that fishery sustainable health development must solve.In the process explored diseases prevention and treatment approach, people recognize the superiority of immune diseases prevention technology gradually, and the research of fish immunology comes into one's own gradually in the world in recent years.
1996, Science magazine was delivered and has been entitled as " natural immunity is to the directive function of Acquired immune response " one literary composition, clearly consequence like this is brought up in the natural immunity first.Current Opinion In Immunology in 1997 publishes the article and proposes pattern recognition receptors (pattern-recognizing receptors, PRRs) relevant with cause of disease molecular pattern (pathogen-associated molecular patterns, PAMPs) concept, the ability of immune system recognition " oneself " " non-own " is given in the pattern recognition effect of proving PRRs, indicates that natural immunity research enters a brand-new stage.Fish are acquired immunity (specific immunity) and the natural immunity (non-specific immunity) and the vertebrates of depositing, but compared with Mammals, fish acquired immunity mechanism goes back imperfection, mainly relies on the natural immunity play a role when resisting pathogenic micro-organism.
Toll-like receptor (Toll-like receptors, TLRs) family is main " pattern recognition receptors " (PRRs) of animal identification invasion pathogenic agent, almost can identify the structural motif " molecular pattern that cause of disease is relevant " (PAMPs) of the high conservative of all pathogenic micro-organisms, startup body generation innate immunity play a part very crucial.PAMPs comprises various bacterial cell wall component, as lipopolysaccharides, polypeptide sugar, teichoic acid etc., and the RNA of virus, the seminose on yeast cells wall, also has flagellin, DNA of bacteria etc.In the mankind verified there is viral recognition function have TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9.Compared with studying with higher mammals such as the mankind, the research of TLRs in fish and other vertebratess such as low is also fewer, but it has been generally acknowledged that TLR1,2,4,5 and 6 mainly participates in the identification of bacterial component, and TLR3,7 and 8 primary specificity ground identify viral composition, TLR9 all can identify both.
Hybridized prussian carp (Carassius auratus gibelio) be utilize triploid upright pond crucian carp for maternal, diplontic Singuo Red Carp be male parent, lure that the ovum of upright pond crucian carp carries out gynogenesis and the crucian cultivation new variety of cultivating into by the method for artificial hybridization, there is due to it features such as fast growth, wide accommodation, meat flavour deliciousness, become one of domestic improved seeds wideling popularize cultivation.In recent years, due to the factor such as deterioration, the expansion of cultivation scale, the lack of standardization of fishing medicine use of water surrounding, hybridized prussian carp disease incidence constantly rises, especially occur in China's hybridized prussian carp main culture zone Jiangsu Province in August, 2011 acute epidemic situation " crucian viral hemorrhagic is sick; " epidemic disease rapid spread in short period of time, onset area is more than 100,000 mu, and mortality ratio, up to 90-100%, causes financial loss to reach several hundred million unit.This large freshwater fish virusology class post scientist Zeng Lingbing researcher of country of research department leads Team Member to get involved rapidly the very first time, be " carp simplexvirus II type " (Cyrinid herpesvirus-2, CyHV-2) by the cause of disease of the technology determination hybridized prussian carp viral hemorrhagic diseases such as pathogen separation, electron microscopic observation, recurrent infection and pcr amplification.Viral hemorrhagic disease due to hybridized prussian carp is Late Cambrian at home, lack relevant early-stage Study basis, and understand less, so there is no effective immune protection technology and methods for the treatment of for this virus disease at present for the immune mechanism of hybridized prussian carp with mechanism.
TLRs is the required molecule of host immune, the pathogenic micro-organism of being invaded by perception, identifies pathogen-associated molecular pattern, signal in active cells, and the natural immune system activating host produces immunne response, and then resists the infection of pathogenic micro-organism.TLR9, as the important member of TLRs family, participates in the immunomodulatory of fish, reflects the immunological competence of fish to a certain extent, therefore can as the index of immunologic surveillance in fish disease control.About the research of hybridized prussian carp TLR9 gene, there is not been reported, this research department utilize RACE technology first from hybridized prussian carp clone obtain the full length cDNA sequence of TLR9 gene, its Genbank sequence number is KC816577.
Real-time fluorescence quantitative PCR (real-time fluorescent quantitative PCR) is the nucleic acid quantitation technique grown up on the basis of Standard PCR technology.Compared with Standard PCR, Real-Time Fluorescent Quantitative PCR Technique have specificity high, highly sensitive, can quantitatively, effectively solve PCR pollution problem and level of automation advantages of higher, ensure that reliability and the repeatability of result, be widely used in the research field such as molecular biology and medical science.Along with the further research and development of real-time fluorescence quantitative PCR detection kit, Real-Time Fluorescent Quantitative PCR Technique have also been obtained and applies widely in aquatic animal disease diagnosis.
Summary of the invention
The object of this invention is to provide a kind of method that fluorescence RT-PCR detects hybridized prussian carp TLR9 gene relative expression quantity.Compared with Standard PCR, the method has highly sensitive, high specificity, time short, reproducible advantage.
To achieve these goals, the technical solution used in the present invention is as follows:
Fluorescence RT-PCR detects a method for hybridized prussian carp TLR9 gene relative expression quantity, and step is as follows:
(1) primer sequence for hybridized prussian carp TLR9 gene design is as follows:
TLR9-qF:5’–TCCAGAGTCATTGGCTGGTGTT–3’
TLR9-qR:5’–CATAGAGATGCTTCAGGAGGGG–3’
According to crucian β-actin gene cDNA sequence (JX413519), design the reference gene Auele Specific Primer being applicable to real-time fluorescence quantitative PCR for a pair and detecting, primer sequence is as follows:
β-actin-qF：5’–CACCACGGCCGAAAGAGAAATT–3’
β-actin-qR：5’–AGGAAGGAAGGCTGGAAGAGAG–3’；
(2) extraction and purification of total serum IgE: adopt conventional RNA Isolation and purification method, extracts the total serum IgE obtaining purifying from hybridized prussian carp spleen;
(3) synthesis of cDNA first chain: with hybridized prussian carp spleen total serum IgE for templated synthesis cDNA first chain;
(4) real-time fluorescence quantitative PCR: adopt Power2 × SYBR Real-time PCR Premixture test kit of Bioteke to carry out.With cDNA first chain of above-mentioned synthesis for template, in above-mentioned (1), TLR9-qF, TLR9-qR and β-actin-qF, β-actin-qR are primer, carry out real-time fluorescence quantitative PCR amplified reaction, 3 parallel pipes established by each sample, and the Ct value that pcr amplification terminates rear gained 3 parallel pipes is taken the mean.
Real-time fluorescence quantitative PCR amplification system is as follows:
Power2 × SYBR Real-time PCR Premixture mixture 25 μ L, 20 μm of ol/L forward primers and each 1 μ L of reverse primer, cDNA the first chain template 2 μ L, aseptic double-distilled water 21 μ l, total reaction volume is 50 μ L.
Real-time fluorescence quantitative PCR reaction parameter arranges as follows:
95℃2min；
95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 20s(totally 40 circulations).
(5) calculating of TLR9 gene relative expression quantity in hybridized prussian carp spleen tissue: after real-time fluorescence quantitative PCR completes, according to the Ct value calculating hybridized prussian carp obtained by the Δ Ct and 2 under time period each after virus infection -(Δ Δ Ct)value, method of calculation are as follows:
ΔCt=Ct（TLR9）-Ct（β-actin）
Δ Δ Ct=Δ Ct(TLR9)-Δ Ct(0 time healthy hybridized prussian carp)
With 2 -(Δ Δ Ct)in numeric representation hybridized prussian carp spleen, TLR9 gene is relative to the expression amount of healthy hybridized prussian carp TLR9 gene.
Compared with prior art, the present invention has the following advantages:
(1) Real-Time Fluorescent Quantitative PCR Technique of the present invention's employing is compared with Standard PCR, and Real-Time Fluorescent Quantitative PCR Technique collects fluorescent signal automatically, avoids the subjectivity that naked eyes judge, can improve the sensitivity of experiment; Real-Time Fluorescent Quantitative PCR Technique adopts totally-enclosed reaction, without the need to PCR aftertreatment, avoids polluting, ensure that the reproducibility and reliability of result; Real-Time Fluorescent Quantitative PCR Technique also eliminates the follow-up tedious steps such as electrophoresis, quantitative scanning in Standard PCR, substantially reduces experimental period.
(2) gene expression abundance of TLR9 gene reflects the immune activity of hybridized prussian carp to a certain extent, when TLR9 gene relative expression quantity increases, prompting hybridized prussian carp may be in pathogen infection state, and does not now also observe on hybridized prussian carp body surface with naked eyes and have any pathogen infection illness.Therefore by the change of TLR9 gene relative expression quantity in monitoring hybridized prussian carp spleen tissue, can learn that whether hybridized prussian carp is by pathogen infection, takes effective prophylactic treatment measure in time ahead of time, avoid the severe development of situation to cause irremediable loss.
(3) TLRs is the required molecule of host immune, the pathogenic micro-organism of being invaded by perception, identifies pathogen-associated molecular pattern, signal in active cells, and the natural immune system activating host produces immunne response, and then resists the infection of pathogenic micro-organism.TLR9 is as the member of TLRs family, and participate in the immunomodulatory of fish, TLR9 identifiable design virus composition, after virus infection host, the expression amount of TLR9 significantly raises.Therefore can be monitored by TLR9 gene pairs virus, once find that expression amount raises, take preventive measures immediately, get the time and do further detailed diagnostics and treatment.
Accompanying drawing explanation
Fig. 1 is hybridized prussian carp TLR9 gene and reference gene β-actin real-time fluorescence quantitative PCR product electrophoresis result;
Swimming lane is from left to right followed successively by DL500DNA Marker, TLR9 gene (140bp), β-actin (198bp), DL500DNA Marker.
The Differential expression analysis that Fig. 2 is TLR9 gene in the hybridized prussian carp spleen tissue of healthy hybridized prussian carp and abdominal injection CyHV-2 virus.
Embodiment
Following Examples further illustrates the present invention, but should not regard limitation of the present invention.
Embodiment 1:
The artificial liver support of hybridized prussian carp:
Collect the viscera tissues such as the liver,spleen,kidney of disease fish (infection of carp simplexvirus II type) in sterile petri dish, shred with Sterile ophthalmic, add the DPBS (Sigma Products) of 10 times of volumes (V/W), to proceed in glass homogenizer and grind to form tissue homogenate under ice bath.Tissue homogenate is transferred to (BD Products) in 50mL centrifuge tube, melts again in room temperature after freezing at 280 DEG C, multigelation like this 3 times, subsequently at 4 DEG C, the centrifugal 30min (Sigma, 3K215) of 4000r/min.Get supernatant liquor to filter through 0.22 μm of filter (Nalgene Products).(the copy number of virus is 1.6 × 10 to the tissue homogenate filtrate obtained 7copies/ μ L) immediately for fish body Experimental infection, residual filtrate can be sub-packed in cryopreservation tube (Corning Products) and save backup at-80 DEG C.
Experimental infection carries out in aquarium, and water body volume is 40L, continuous charge, water temperature 25 DEG C.Get healthy hybridized prussian carp (the long 10 ~ 15cm of body) 60 tails not using vaccine in the recent period, every tail is viral suspension on abdominal injection 0.2mL, as infection group and viral infection group; With DPBS injection as a control group, continue to cultivate in aquarium, get its spleen tissue respectively at 6h, 12h, 24h, 48h, the 72h after injecting virus, be placed in rapidly the frozen preservation of liquid nitrogen.
Embodiment 2:
The Isolation and purification of total serum IgE:
(1), after the spleen tissue 50-100mg getting liquid nitrogen cryopreservation adds 1mL Trizol, with the thorough homogenate mixing of homogenizer, room temperature places 5min, makes its abundant cracking.
(2) 4 DEG C, 12,000r/min, centrifugal 10min, supernatant is transferred to one new in the 1.5mL centrifuge tube of RNase, abandons precipitation.
(3) often pipe adds 200 μ L chloroforms, and after vibration mixing, room temperature places 15min.
(4) 4 DEG C, 12,000r/min, centrifugal 15min, draw upper strata aqueous phase, is transferred to one new in the 1.5mL centrifuge tube of RNase.
(5) often pipe adds 0.5mL Virahol, and gently after mixing, room temperature places 5-10min.
(6) 4 DEG C, 12,000r/min, centrifugal 10min, abandons supernatant, and RNA is sunken at the bottom of pipe.Often pipe adds 75% ethanol of 1mL DEPC water preparation, and gentle vibration centrifuge tube, suspend precipitation.
(7) 4 DEG C, 8,000r/min, centrifugal 5min, abandons supernatant as far as possible, and gained precipitation is the RNA of hybridized prussian carp spleen tissue, after room temperature dries 5-10min, adds DEPC water dissolution, and saves backup in-80 DEG C.
Embodiment 3:
The real-time fluorescence quantitative PCR of TLR9 gene detects:
(1) primer:
TLR9-qF:5’–TCCAGAGTCATTGGCTGGTGTT–3’
TLR9-qR:5’–CATAGAGATGCTTCAGGAGGGG–3’
The PCR primer estimated length of hybridized prussian carp TLR9 gene is 140bp, product length after agarose gel electrophoresis result display pcr amplification is consistent with estimated length, and be single band, the primer designed by explanation has very strong specificity, is suitable for real-time fluorescence quantitative PCR and detects.Agarose gel electrophoresis result as shown in Figure 1.
Simultaneously according to crucian β-actin gene cDNA sequence (JX413519) found in Genbank, design the reference gene Auele Specific Primer being applicable to real-time fluorescence quantitative PCR for a pair and detecting, primer sequence is as follows:
β-actin-qF：5’–CACCACGGCCGAAAGAGAAATT–3’
β-actin-qR：5’–AGGAAGGAAGGCTGGAAGAGAG–3’；
The PCR primer estimated length of hybridized prussian carp β-actin gene is 198bp, product length after agarose gel electrophoresis result display pcr amplification is consistent with estimated length, and be single band, the primer designed by explanation has very strong specificity, is suitable for real-time fluorescence quantitative PCR and detects.Agarose gel electrophoresis result as shown in Figure 1.
(2) synthesis of cDNA first chain:
The hybridized prussian carp spleen total serum IgE obtained using embodiment 2 is as template, Oligo(dT with in ImProm-II TM Reverse Transcription box (Promega)) as primer, carry out reverse transcription according to test kit specification sheets, synthesis cDNA first chain, the cumulative volume of reaction system is 20 μ L.
(3) real-time fluorescence quantitative PCR:
Real-time fluorescence quantitative PCR adopts Power2 × SYBR Real-time PCR Premixture test kit of Bioteke to carry out.With cDNA first chain of synthesis in above-mentioned (2) for template, in above-mentioned (1), TLR9-qF, TLR9-qR and β-actin-qF, β-actin-qR are primer, carry out real-time fluorescence quantitative PCR amplified reaction, 3 parallel pipes established by each sample, and the Ct value that pcr amplification terminates rear gained 3 parallel pipes gets its mean number.
Real-time fluorescence quantitative PCR amplification system is as follows:
Power2 × SYBR Real-time PCR Premixture mixture 25 μ L, 10 μm of ol/L forward primers and each 1 μ L of reverse primer, cDNA the first chain template 2 μ L, aseptic double-distilled water 21 μ l, total reaction volume is 50 μ L.
Real-time fluorescence quantitative PCR reaction parameter arranges as follows:
95℃2min；
95 DEG C of 15s, 60 DEG C of 15s, 72 DEG C of 20s(totally 40 circulations).
(4) calculating of TLR9 gene relative expression quantity in the hybridized prussian carp spleen of virus infection:
After real-time fluorescence quantitative PCR completes, calculate hybridized prussian carp by the Δ Ct and 2 under time period each after virus infection according to obtained Ct value -(Δ Δ Ct)value, method of calculation are as follows:
ΔCt=Ct（TLR9）-Ct（β-actin）
Δ Δ Ct=Δ Ct(TLR9)-Δ Ct(0 time healthy hybridized prussian carp)
With 2 -(Δ Δ Ct)in numeric representation virus infection hybridized prussian carp spleen, TLR9 gene is relative to the expression amount of healthy hybridized prussian carp TLR9 gene.
2 -(Δ Δ Ct)numerical value to represent in virus infection hybridized prussian carp spleen TLR9 gene relative to the expression multiple of healthy hybridized prussian carp TLR9 gene.β-actin i.e. beta-actin, it is the important skelemin of one of cell, all express in all types of cell, and its expression level is only by promotor and RNA polymerase is interactional affects, factor affected by environment is very little, expression level is comparatively constant, and therefore β-actin quilt is as the reference gene of generally acknowledging, the conventional internal reference done when measuring target gene expression amount.In this experiment 2 -(Δ Δ Ct)numerical value is larger, shows that the expression amount of TLR9 gene is higher.
Fig. 2 is the relative expression quantity of TLR9 gene in the different time sections hybridized prussian carp spleen tissue after virus infection.As can be seen from the figure, the expression amount of the 12h after virus infection, TLR9 gene just starts to raise, and expression amount is 10.1 times of healthy hybridized prussian carp (0h); Metainfective 24h, TLR9 expression amount is 51.3 times of healthy hybridized prussian carp; During 72h, TLR9 expression amount reaches the highest, is 75.7 times of healthy hybridized prussian carp.Above result shows, when TLR9 gene relative expression quantity increases, namely show that hybridized prussian carp has been in pathogen infection state, immunity system intrinsic in hybridized prussian carp body starts, although now do not observe from naked eyes the illness that hybridized prussian carp body surface has any infection.
The Ct value of TLR9 gene of table 1 for recording in different time sections
As can be seen from Table 1, the standard deviation of 12h, 24h, 48h, 72h gained Ct value of TLR9 gene after virus infection is all very little, illustrates that this method has extraordinary repeatability.
Therefore, the gene expression abundance of TLR9 gene reflects the immune activity of hybridized prussian carp to a certain extent, can by the change of TLR9 gene relative expression quantity in monitoring hybridized prussian carp spleen tissue, find whether hybridized prussian carp has infected cause of disease ahead of time, take effective prophylactic treatment measure in time, avoid the severe development of situation to cause irremediable massive losses.
SEQUENCE LISTING
 
<110> Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
 
<120> fluorescence RT-PCR detects the method for hybridized prussian carp TLR9 gene relative expression quantity
 
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Claims (1)

1. the Auele Specific Primer of fluorescence RT-PCR detects the application infected in carp simplexvirus II type hybridized prussian carp indicator in preparation, and described primer is TLR9-qF:5 ' – TCCAGAGTCATTGGCTGGTGTT – 3 '; TLR9-qR:5 ’ – CATAGAGATGCTTCAGGAGGGG – 3 '; Internal reference: β-actin-qF:5 ’ – CACCACGGCCGAAAGAGAAATT – 3 '; β-actin-qR:5 ’ – AGGAAGGAAGGCTGGAAGAGAG – 3 '.
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CN101796932A (en) * 2010-04-07 2010-08-11 江苏海辰科技集团 Method for optimal seedling selection and breeding of novel carassius auratus gibelio series
CN101919362A (en) * 2010-08-31 2010-12-22 中国水产科学研究院淡水渔业研究中心 Semi-artificial breeding technology for Carassius aurutus gibelio

Patent Citations (2)

* Cited by examiner, † Cited by third party
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