CN104975093A - Application of serum ssc-miR-215 as molecular marker on detecting stress injury of intestinal tract of pigling - Google Patents

Abstract

The invention belongs to the field of biology, and relates to an application of serum ssc-miR-215 as a molecular marker on detecting stress injury of the intestinal tract of a pigling. A nucleotide sequence of serum ssc-miR-215 is represented as SEQ ID NO.1. A nucleotide sequence of a stem-loop primer required by reverse transcription of ssc-miR-215 is represented as SEQ ID NO.2. A nucleotide sequence of a forward primer required by augmentation of ssc-miR-215 is represented as SEQ ID NO.3. Diagnosis of stress injury of the intestinal tract of a weaned pigling is realized by adopting the above primers. A diagnostic method comprises extraction of serum total RNA, separation of small molecular RNA, synthesis of cDNA, and augmentation and authentication of PCR. The invention provides guidance for prevention, diagnosis and treatment of stress injury of the intestinal tract of a pigling, and provides a novel molecular marker for marker assisted selection of an anti-stress pig line.

Description

Serum ssc-miR-215 is as the application detecting intestine of young pigs stress damage molecular marked compound

Technical field

The invention belongs to domestic animal gene engineering technology field, be specifically related to serum ssc-miR-215 as the application detecting intestine of young pigs stress damage molecular marked compound.

Background technology

Stress of baby pigs caused ablaction is a great problem in whole world pig-breeding, all causes huge financial loss to pig industry every year.The grice diarrhoea that stress cause, the especially the most common a kind of stress disease maximum with hazardness in current Swine Production.The main target spot of ablactation stress is the gi tract of piglet, wherein particularly serious to the damage of small intestine.The Small Intestine of Piglets tissue injury that ablactation stress causes and reconstruction have had a strong impact on growth and the maturation of intestine of young pigs, thus constrain health and the growth of piglet.Equally, ablactation stress initiation intestinal tract injury causes the decline of stomach and intestine enzymic synthesis and receptivity to be considered to the primary factor (Wu Xiaoxiong, 1999) of diarrhea of weaned piglets; Intestinal mucosa is impaired is the one of the main reasons (Yan Hongxiang etc., 2006) causing grice diarrhoea.Therefore weaning period is considered to an important period of intestinal growth, the enteron aisle in this period due to piglet is not yet reached maturity, and ablactation stress likely causes lifelong impact (Smith et al, 2010) to intestine of young pigs health.

At present, enteron aisle the strongest impaired direct evidence carries out the detection of intestinal mucosa morphology change after being butchered by piglet.The reduction of villi height and the increase of Crypt depth confirm the topmost index (Nabuurs etc., 1993) that mucosal structure is impaired in research report.But, butcher the elaboration that destructive research can only be used for research mechanism.If by non-mode of butchering, such as find the index of reflection enteron aisle stress damage reliably in blood, for prevention as early as possible, Diagnosis and Treat enteron aisle stress damage, raising Swine Production performance, increase economic benefit of raising pigs, and particularly it can be used as molecular marked compound to be applied in pig molecular breeding to improve live pig holistic health level, all there is very important production practice meaning and scientific meaning.Therefore, scientists has carried out correlative study (Qian Zhongcang etc., 2009 to the change of diamine oxidase (DAO) activity, D-ALPHA-Hydroxypropionic acid content and Determination of cortisol etc. in weanling pig serum/plasma; Clock arms etc., 2013; Hu Caihong etc., 2013; Huang Qichun etc., 2014; Wolvekamp etc., 1994; Li Junyou etc., 2000; Hu Quanzhou, 2007).But the detection of These parameters change is relative value, requires corresponding control sample and do reference and just can reach a conclusion by carrying out significant difference analysis to data, therefore do not possess diagnostic value.

The discovery of serum/plasma miRNA serum s is undoubtedly an important breakthrough in miRNAs research field, for the research of miRNAs on animal provides new Research Thinking.Existing research confirms that serum miRNAs not only exists and has popularity, stability and specificity, particularly has the unrivaled advantages such as non-invasive and convenience.In addition, the growing of its change and body, disease development and multiple physiological phenomenon stress be waited closely related.Mitchell etc. (2008) first confirm the existence of miRNAs in tumour patient blood plasma.Almost simultaneously, Chen etc. again at Diseases such as healthy human, lung cancer, colorectal carcinoma and diabetes and comprise in the animal serums such as mouse, ox and horse and blood plasma and detect multiple miRNAs, and confirm miRNAs in serum and plasma without significant difference and its content hardly by the impact of room temperature, high temperature, pH, thawing.Gilad etc. (2008), by comparing the miRNAs in gravid woman's serum, urine, saliva, amniotic fluid and Pleural fluid, think that serum miRNAs is more suitable for as a kind of new biomarker thing.Blondal etc. (2013) point out that miRNAs can be discharged into body fluid from tissue fast when body generation pathological change.Prompting serum circulates miRNAs has in time, the advantage of specificity reflection body physiological state.Have been reported and show that the detection of serum/plasma miRNA serum s contributes to diagnostic organization organ damage (Redell etc., 2010), sacroiliitis (Murata etc., 2010), cardiovascular system diseases (Tijsen etc., 2012), hepatitis (Zhang etc., 2012) and diabetes (Guay etc., 2013) various diseases such as, more horn of plenty (Liu etc., 2013 of the achievement in research particularly in various malignant tumour; Madhavan etc., 2013; Sozzi etc., 2014).

Summary of the invention

Object of the present invention, for the deficiencies in the prior art, provides the application of serum ssc-miR-215 as detecting intestine of young pigs stress damage molecular marked compound.

The object of the invention is to be achieved through the following technical solutions:

Serum ssc-miR-215 is as the application detecting intestine of young pigs stress damage molecular marked compound, and the nucleotide sequence of described ssc-miR-215 is as shown in SEQ ID NO.1.

Further, the reverse transcription primer (stem-ring primer) of described ssc-miR-215 is as shown in SEQ ID NO.2.

Further, described ssc-miR-215 forward primer is as shown in SEQ ID NO.3, and ssc-miR-215 reverse primer is as shown in SEQ ID NO.4.

Above-mentioned application comprises the following steps:

(1) collect weanling pig whole blood, prepare serum sample;

(2) Plasma/Serum Exosome Kit (Norgen, 49200) reagent is adopted to extract total RNA in serum sample;

(3) the serum small molecular RNA in miRNeasy Mini Kit (Qiagen 217004) test kit extraction total RNA is adopted;

(4) the ssc-miR-215 reverse transcription primer as shown in SEQ ID NO.2 is adopted to carry out reverse transcription to microRNA, obtain cDNA: described reverse transcription system is: microRNA sample 2.5ul (40ng/ul), ssc-miR-215 reverse transcription primer 0.5ul (2uM), dNTP 0.25ul (10mM each), 5 × RT buffer 1.0ul, RNase inhibitor 0.25ul (40U/ul), M-MLV 0.5ul (200U/ul), reaction system cumulative volume is 5.0ul, mixed uniformly reaction system is placed 42 DEG C of reaction 60min, place 70 DEG C of reaction 15min again, 4 DEG C of preservations,

(5) take U6 as internal reference, adopt the forward primer of ssc-miR-215 and reverse primer to carry out quantitative fluorescent PCR to cDNA; Reaction system is ssc-miR-215 forward primer 0.4ul (10uM) shown in the cDNA 1.0ul that step 4 obtains, 2 × SYBR Green Mix10.0ul, RNase-free Water 8.2ul, SEQ ID NO.3, ssc-miR-215 reverse primer 0.4ul (10uM) shown in SEQ ID NO.4, reaction system cumulative volume is 20.0ul; Quantitative fluorescent PCR reaction conditions: 50 DEG C of reaction 2min, 95 DEG C of reaction 5min; Then 95 DEG C of reaction 15s, 60 DEG C of reactions 15s, 40cycles.

(6) if fluorescent quantitative PCR result Δ Ct value negative value, then weanling pig enteron aisle stress damage is shown; If fluorescent quantitative PCR result Δ Ct value be on the occasion of, then show that weanling pig enteron aisle stress damage does not occur.

Beneficial effect of the present invention is: the present invention has filled up the blank of current weanling pig enteron aisle stress damage diagnostic method, provides serum ssc-miR-215 as the application detecting intestine of young pigs stress damage molecular marked compound.The invention has the advantages that: the present invention makes public for the first time and adopts serum ssc-miR-215 and be respectively used to reverse transcription and the amplification stem-ring primer of ssc-miR-215 and forward primer, reference gene is that U6 stress diagnose the enteron aisle of weanling pig, if diagnostic result Δ Ct value result negative value, then show weanling pig enteron aisle stress damage, Δ Ct value be on the occasion of, then show that weanling pig enteron aisle stress damage does not occur.The excavation of this marker and be applied as the prevention of weanling pig enteron aisle stress damage, Diagnosis and Treat provides guidance, especially the marker assisted selection of pig stress-resistant line provides a new molecular marked compound.

Accompanying drawing explanation

Fig. 1 is No. 3 Jejunum of Piglets fluff morphology figure;

Fig. 2 is 5 Jejunum of Piglets fluff morphology figure;

Fig. 3 is No. 13 Jejunum of Piglets fluff morphology figure;

Fig. 4 is 15 Jejunum of Piglets fluff morphology figure.

Embodiment

Embodiment 1: the present embodiment passes through serum microRNA and the enteron aisle microRNA correlation analysis of weanling pig, obtains ssc-miR-215, comprises the following steps:

1. intestine of young pigs sample Total RNAs extraction

From parity and date of birth all identical 4 nests, 25 age in days piglets, choose 24 (6, every nests) is research object, is equally divided into lactation group and wean group (wean on the same day).1d, 4d and 7d after wean group weaned piglet respectively, often organizes and butchers piglet 4 (1, every nest).Collect jejunal tissue samples, adopt Trizol method to extract intestine of young pigs total tissue RNA sample.

2. microRNA library construction

4 of each treatment group biology are repeated total serum IgE sample and forms RNA sample pond respectively by carrying out balanced mix after concentration conversion, utilize the denaturing polyacrylamide gel electrophoresis of 15% from total serum IgE sample, be separated microRNA within the scope of 20-30nt, connection 5 ' and 3 ' joint, purifying, reverse transcription is cDNA and forms sequencing library after pcr amplification.Purifying microRNA library, upper machine order-checking after quality examination is qualified.This experiment adopts the high-flux sequence of the single-ended 50bp order-checking Pattern completion sample of Illumina Hiseq2000 order-checking platform.The raw data that order-checking obtains need remove primer and adaptor sequence, and the quality test passed through sequenced fragments base and length screening, the final sequenced fragments selecting reliable in quality.

3. the analysis of weanling pig enteron aisle differential expression miRNAs

After miRNAs expression amount is normalized, carry out Differential expression analysis result to the miRNAs identified to show (table 1 ~ table 3): after weaned piglet, 1d group is compared with its control group, find the miRNAs of 16 differential expressions altogether, wherein 11 are significantly raised, and significantly lower for 5; After wean, 4d group is compared with its control group, finds the miRNAs of 98 differential expressions altogether, and wherein 92 are significantly raised, and significantly lowers for 6; After wean, 7d group is compared with its control group, finds the miRNAs of 22 differential expressions altogether, and wherein 15 are significantly raised, and significantly lowers for 7.Therefore, ablactation stress significantly affects the expression of miRNAs in intestine of young pigs tissue, and 4d impact is the most remarkable after wean, be one of minority downward miRNAs in maximum gene expression abundance in the miRNAs that expresses at all differences of miR-215 and miR-194b in addition.Therefore, follow-up microRNA chip and fluorescent quantitative PCR experiment mainly compare the difference of age groups on the 4th and control group thereof after weaned piglet.

Table 1: wean latter 1 day age group

Table 2: wean latter 4 days age groups

Table 3: wean latter 7 days age groups

Two, microRNA chip obtains the differential expression spectrum of weanling pig serum microRNA

1. piglet serum sample Total RNAs extraction

Test piglet 2h before butchering, adopt 5ml syringe collecting whole blood, the centrifugal 10min of 3000g, prepares serum sample.Plasma/Serum Exosome Kit (Norgen, 49200) reagent is adopted to extract Total RNA in serum sample.4 biology repetition total serum IgE samples of wean latter 4 days age groups and control group thereof are formed RNA sample pond, for follow-up array experiment respectively by carrying out balanced mix after concentration conversion.

2.microRNA array experiment

Microarray Experiments is undertaken by LC Sciences company.Adopt 5ug total serum IgE sample, use Poly (A) polysaccharase to add poly (A) tail at total serum IgE 3' end, then an oligonucleotide mark is connected (ligation) for follow-up fluorescent mark with this poly (A) tail.Hybridization utilizes the Circulation of micro circulation pump (AtacticTechnologies) to spend the night on μ Paraflo micro-fluid chip to carry out.Detection probes is from newfound miRNA sequence in all ripe miRNA sequence of pig in miRbase20.0 database and high-flux sequence.Detection probes all uses PGR (photogenerated reagent) chemical method to carry out fabricated in situ.Hybridization melting temperature(Tm) is balanced by chemically modified detection probes.Hybridization uses the 100 μ L 6xSSPE damping fluids (0.90M NaCl, 60mM Na2HPO4,6mM EDTA, pH 6.8) containing 25% methane amide, and hybridization temperature is 34 DEG C.After RNA and probe hybridization, to flow in micro-fluid chip cocycle with the Cy3 dyestuff of mark specific combination and dye.

3. the analysis of weanling pig serum differential expression miRNA

Utilize laser scanner (GenePix 4000B, Molecular Device) to gather hybridization image and use Array-Pro image analysis software (Media Cybernetics) to carry out image digitazation conversion.First data analysis is subduction background value, then uses LOWESS to filter (Locally-Weighted Regression) and carries out signal normalization.Statistical study setting P<0.01, signal<500.Carry out analytical results to chip data to show: 115 miRNAs are differential expression (table 4) in weanling pig serum; The hybridization signal of 64 miRNAs is less than 500, and these miRNAs can think and not express in piglet serum; The difference of expression in weanling pig and control group thereof of 122 miRNAs is not remarkable.

Table 4

Three, the consistency analysis of microRNAs differential expression change in weanling pig enteron aisle and serum

Data in table 1 ~ 4 are contrasted, find that the miRNAs all in differential expression in weanling pig enteron aisle and serum has 33, wherein have the expression change direction of 16 miRNAs consistent, be both up-regulated or downward, the expression change direction of other 17 miRNAs is inconsistent.For avoiding false positive results, │ log2 │ >=1.5 are expressed the consistent miRNAs of change to 16 and are screened with │ log2 │ >=2 of weanling pig and control group thereof in serum miRNA chip results and in high-flux sequence result further, find that only ssc-miR-215 and ssc-miR-194b meets screening conditions, and miR-215 and miR-194b expresses in most high abundance in intestine of young pigs, is minority lowers one of miRNAs in weanling pig enteron aisle.

Embodiment 2: the ssc-miR-215 that embodiment 1 obtains is used for marking weanling pig enteron aisle stress damage by the present embodiment.

(1) random selecting 24 weanling pigs, are labeled as 1 ~ No. 24 respectively, collect weanling pig whole blood, prepare 1 ~ No. 24 serum sample;

(2) Plasma/Serum Exosome Kit (Norgen, 49200) reagent is adopted to extract total serum IgE in serum sample;

(3) the serum microRNA in miRNeasy Mini Kit (Qiagen 217004) test kit extraction Total RNA is adopted;

(4) the ssc-miR-215 reverse transcription primer as shown in SEQ ID NO.2 is adopted to carry out reverse transcription to microRNA, obtain cDNA: described reverse transcription system is: microRNA sample 2.5ul (40ng/ul), ssc-miR-215 reverse transcription primer 0.5ul (2uM), dNTP 0.25ul (10mM each), 5 × RT buffer 1.0ul, RNase inhibitor 0.25ul (40U/ul), M-MLV 0.5ul (200U/ul), reaction system cumulative volume is 5.0ul, mixed uniformly reaction system is placed 42 DEG C of reaction 60min, place 70 DEG C of reaction 15min again, 4 DEG C of preservations,

(5) with U6 (the reverse transcription primer ATGCGGCGGCGTATTGT of U6, the forward primer GACCTCGCGGTGGTTATG of U6, the reverse primer ATGCGGCGGCGTATTGT of U6) be internal reference, adopt the forward primer of ssc-miR-215 and reverse primer to carry out quantitative fluorescent PCR to cDNA; Reaction system is ssc-miR-215 forward primer 0.4ul (10uM) shown in the cDNA 1.0ul that step 4 obtains, 2 × SYBR Green Mix 10.0ul, RNase-free Water 8.2ul, SEQ ID NO.3, ssc-miR-215 reverse primer 0.4ul (10uM) shown in SEQ ID NO.4, reaction system cumulative volume is 20.0ul; Quantitative fluorescent PCR reaction conditions: 50 DEG C of reaction 2min, 95 DEG C of reaction 5min; Then 95 DEG C of reaction 15s, 60 DEG C of reactions 15s, 40cycles;

(6) fluorescent quantitative PCR result represents with Δ Ct value: namely in same sample, and Δ Ct=Ct object Ji is Yin – Ct reference gene.If Δ Ct value negative value, then show weanling pig enteron aisle stress damage; If fluorescent quantitative PCR result Δ Ct value be on the occasion of, then show that weanling pig enteron aisle stress damage does not occur.

The fluorescent quantitative PCR result of table 5 weanling pig serum ssc-miR-215

Label	1	2	3	4	5	6	7	8
									ΔCt	-1.58	2.77	0.75	1.072	-0.25	-1.35	2.25	-1.19
Label	9	10	11	12	13	14	15	16
									ΔCt	-0.47	0.76	2.01	1.32	0.29	-3.33	-0.53	-1.33

Label	17	18	19	20	21	22	23	24
									ΔCt	1.58	-0.75	2.77	-1.47	1.07	-1.20	2.08	-1.75

Choose │ Δ Ct │ minimum No. 3, No. 5, No. 13 and No. 15 piglets are butchered, collect jejunal tissue, carry out the making of jejunum Section for light microscopy.As shown in Fig. 1-4 and table 6.

Table 6 butchers jejunum villi height and the Crypt depth of piglet

	3	5	13	15
					Height of naps, μm	426.50	213.50	421.80	218.85
Crypt depth, μm	110.47	176.49	161.67	218.02

As can be seen from Fig. 1-4 and table 6, No. 3 and No. 13 Jejunum of Piglets fluff morphology structural integrities, edge clear between villus epithelial cells, and No. 5 and No. 15 weanling pig jejunum villi obviously shorten, villus epithelial cells obscure boundary is clear, and Intraepithelial goblet cell quantity increases, and crypts is deepened, No. 5, susceptible of proof and No. 15 piglets are subject to ablactation stress, cause intestinal tract injury.The above results shows that the method for the invention has high accuracy and sensitivity.

SEQUENCE LISTING

 

<110> Zhejiang Academy of Agricultural Science

 

<120> serum ssc-miR-215 is as the application detecting intestine of young pigs stress damage molecular marked compound

 

 

<160> 4

 

<170> PatentIn version 3.3

 

<210> 1

<211> 21

<212> DNA

<213> synthetic

 

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augaccuaug aauugacaga c 21

 

 

<210> 2

<211> 50

<212> DNA

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<400> 2

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgtctgt 50

 

 

<210> 3

<211> 20

<212> DNA

<213> synthetic

 

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ccagcgtgat gacctatgaa 20

 

 

<210> 4

<211> 21

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<400> 4

cagtgcaggg tccgaggtat t 21

 

Claims (4)

1. serum ssc-miR-215 is as the application detecting intestine of young pigs stress damage molecular marked compound, and it is characterized in that, the nucleotide sequence of described ssc-miR-215 is as shown in SEQ ID NO.1.

2. application according to claim 1, is characterized in that, the reverse transcription primer (stem-ring primer) of described ssc-miR-215 is as shown in SEQ ID NO.2.

3. application according to claim 1, is characterized in that, described ssc-miR-215 forward primer is as shown in SEQ ID NO.3, and ssc-miR-215 reverse primer is as shown in SEQ ID NO.4.

4. application according to claim 1, is characterized in that, this application comprises the following steps:

(1) collect weanling pig whole blood, prepare serum sample;

(2) Plasma/Serum Exosome Kit reagent is adopted to extract total RNA in serum sample;

(3) microRNA in miRNeasy Mini Kit test kit extraction total RNA is adopted;

(4) the ssc-miR-215 reverse transcription primer as shown in SEQ ID NO.2 is adopted to carry out reverse transcription to microRNA, obtain cDNA: described reverse transcription system is: microRNA sample 2.5ul(40ng/ul), ssc-miR-215 reverse transcription primer 0.5ul(2uM), dNTP 0.25ul (10mM each), 5 × RT buffer 1.0ul, RNase inhibitor 0.25ul (40 U/ul), M-MLV 0.5ul (200 U/ul), reaction system cumulative volume is 5.0ul, mixed uniformly reaction system is placed 42 DEG C of reaction 60min, place 70 DEG C of reaction 15min again, 4 DEG C of preservations,

(5) take U6 as internal reference, adopt the forward primer of ssc-miR-215 and reverse primer to carry out quantitative fluorescent PCR to cDNA; Reaction system is ssc-miR-215 forward primer 0.4ul (10 uM) shown in the cDNA 1.0ul that step 4 obtains, 2 × SYBR Green Mix 10.0ul, RNase-free Water 8.2ul, SEQ ID NO.3, ssc-miR-215 reverse primer 0.4ul (10 uM) shown in SEQ ID NO.4, reaction system cumulative volume is 20.0ul; Quantitative fluorescent PCR reaction conditions: 50 DEG C of reaction 2 min, 95 DEG C of reaction 5 min; Then 95 DEG C of reaction 15 s, 60 DEG C of reactions 15 s, 40cycles;

(6) if fluorescent quantitative PCR result Ct value negative value, then weanling pig enteron aisle stress damage is shown; If fluorescent quantitative PCR result Ct value be on the occasion of, then show that weanling pig enteron aisle stress damage does not occur.