CN105316409A - Specific primers and typing method of class II MHC (major histocompatibility complex) genes for antibacterial potential detection of alligator sinensis - Google Patents
Specific primers and typing method of class II MHC (major histocompatibility complex) genes for antibacterial potential detection of alligator sinensis Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims description 12
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- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 title description 5
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Abstract
The invention relates to a pair of single-locus specific primers for amplifying class II major histocompatibility complex genes for antibacterial potential detection of the alligator sinensis and a typing method, wherein the specific sequences of the primers are as shown in SEQ ID NO. 1-SEQ ID NO. 2. The amplification primers disclosed by the invention are stable in single-locus specific amplification capacity in an alligator sinensis population; a product obtained from amplification can conduct genotyping on various individuals through a subsequent single strand conformation polymorphism (SSCP) experiment, so that the polymorphism of the class II MHC genes of alligator sinensis individuals in the population is assessed and the potential of the alligator sinensis individuals in resisting bacterial diseases is detected; therefore, an important reference is provided for such operations of new population founder selection, reintroduction individual selection, artificial mating selection and the like in alligator sinensis protection work.
Description
Technical field
The present invention discloses unit point Auele Specific Primer and the classifying method of II class MHC (majorhistocompatibilitycomplex, the MHC) gene order that a pair is detected for the Chinese alligator antibacterium potentiality that increase.
Background technology
Chinese alligator (Alligatorsinensis, Fauvel) reptilia is belonged to, Crocodilia, Chinese alligator section, alligator subfamily, Chinese alligator belongs to, it is a kind of ancient animal, originate from the Triassic period before 200,013,000 years, be called as " living fossil " in Reptilia, for the peculiar rare species of China, country-level focused protection animal (1972), annex I is put in " endangered animal and plant international trade pact " (CITES), be classified as critically endangered species by World Conservation Union (IUCN) simultaneously, be be on the hazard in existing crocodile class maximum, one of species the most in imminent danger.The quantity of current Wild Chinese alligator only about 100.
Before about 2.3 hundred million years, the ancestors of Chinese alligator live in same environment with earth overlord dinosaur once.But powerful dinosaur is mysterious before 6,500 ten thousand to disappear, and crocodile has but survived in miraculously, and procreation so far.As a kind of ancient Reptilia, Chinese alligator is the many features that form or behavior all maintain reptiles ancestors, is species indispensable in petrifactology, the research of species evolutionary process.
MHC (majorhistocompatibilitycomplex, MHC) for being present in one group of linked gene group of vertebrates specific chromosome regions, play a key effect in vertebrate immune system, the immunocyte surface receptor abundant species of its coding is antigen-presenting and the vital role factor regulated in body immune response process in immunity system.According to the difference of institute's antigen-presenting type, MHC can be divided into two large classes: I class and II class, wherein the antigen binding domain of albumen coded by II class mhc gene mainly identifies and in conjunction with the polypeptide of exogenous antigen (as bacterium), and by it in passing CD4+T cell, and start follow-up immune response.Therefore, the antigen binding domain (exon 2) of II class MHC generally has height polymorphism, and the individuality that II class MHC antigen binding domain polymorphism is high better can resist bacteriosis.Protection worker is when carrying out newly-built population and selecting the work such as founder and the strong individuality of reintroduction selection adaptation ability; scientifically can select according to the polymorphism height of mhc gene; when stable breeding population manually matches; can also select that there is not homoallelic individuality match thus improve the polymorphism of filial generation, the enhanser resistibility of generation to bacteriosis.
In existing Chinese alligator MHC document, are all the site intersection amplifications utilizing universal primer to carry out mhc gene, its result can only be used for simple evolutionary analysis.The II class MHC primer that the present invention relates to is all through great many of experiments repeated authentication; can not only increase smoothly in Chinese alligator population; show stable unit point specific amplification ability; also by follow-up single strand conformation polymorphism (SSCP), gene type is carried out to Chinese alligator II class mhc gene; effective help we assess II class MHC polymorphism in population, this to Chinese alligator protection work bring great convenience undoubtedly.
Summary of the invention
The object of the invention is to provide primer and the classifying method thereof of the II class mhc gene sequence that a pair is detected for the Chinese alligator antibacterium potentiality that increase.
A pair unit point Auele Specific Primer for the Chinese alligator II class mhc gene that increases, the concrete sequence of primer is:
Beta1085E2F:CTCTGCCCAGTAGGAGCCGTGC (as shown in SEQIDNO:1);
Beta1085E2R:AACAAAGACCCCAGGAATCCAG (as shown in SEQIDNO:2).
The invention also discloses the classifying method of the II class mhc gene that a kind of Chinese alligator antibacterium potentiality detect, namely above-mentioned primer is adopted, under specific amplification condition, carry out target fragment amplification by 10 μ LPCR systems, then utilized by amplified production SSCP to carry out gene type respectively.
10 described μ LPCR systems are: Chinese alligator genomic dna 0.8 μ L, each 0.2 μ L of upstream and downstream primer, ultrapure water 4.4 μ L, 2 × UltraTaqMasterMix4.4 μ L.
The PCR response procedures of described amplimer is: 95 DEG C of denaturations 5 minutes, 95 DEG C of sex change 30 seconds, and 63 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 34 circulations, last 72 DEG C of extensions 10 minutes.
After PCR terminates, all use the sepharose of 1% to add DL2000marker and carry out electrophoresis detection to PCR primer, qualified product deposits in 4 DEG C of refrigerators.
The expanding fragment length of described primer is 364bp.
Describedly the method for single strand conformation polymorphism (SSCP) gene type is utilized to be to Chinese alligator II class MHC sequence specific site:
1) with glass washing composition cleaning size sheet glass, with alcohol swab wiping 3 times after draining, and treat alcohol volatilization post-erection plate, spacer bar is placed between size sheet glass, and with special frame, both sides is clamped, be fixed on mould;
2) prepare the non-denaturing polyacrylamide gel glue of 12%, slowly inject between size sheet glass after glue is mixed, confirm that bubble-free inserts comb, normal temperature horizontal after existing;
3) after gelling is solid, unload also frame from mould and, on electrophoresis apparatus, use agarose sealing; Take off comb, and with filling the irrigation with syringe loading wells of 0.5 × TBE; Put into electrophoresis chamber, pour 0.5 × TBE into, prerunning 20min;
4) sample electrophoresis, draws 7uLPCR product, adds 4uL2 × loadingbuffer, quick centrifugal mixing, be placed on ice rapidly, and add in time in loading wells with rifle, 4 DEG C, 35W, electrophoresis 6-7h after 95 DEG C of sex change 4min30s;
5) glue is unloaded between sheet glass, add 500mL stationary liquid, on shaking table, fix 30min, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
6) add 500mL silver dye liquor, on shaking table, silver dye 30min, outwells silver-colored dye liquor, uses 500mL distilled water to wash rapidly glue 4 times;
7) add the developing solution of 500mL precooling, in shaking table development, wait for that band removes developing solution after clear, add 500mL stationary liquid and stop development, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
8) read band, determine genotype.
Adopt technique scheme, effectively can increase and obtain Chinese alligator mhc gene seat, and can not intersect increase different seat allelotrope or generation amorphs.Mhc gene somatotype can be carried out to Different Individual in Chinese alligator different groups, the same group simultaneously; its amplification obtains product can detect (singlestrandconformationpolymorphism by follow-up single strand conformation polymorphism; SSCP) experiment carries out gene type to different individualities; realize the assessment to the individual II class mhc gene polymorphism of Chinese alligator in population; detect the potentiality of its opposing bacteriosis, for the new population founder selection in Chinese alligator protection work, open countryization are put and returned the work such as individual selection and artificial pairing to provide important references.In addition, adopt the technical program, when relating to sample number and being more, SSCP somatotype can be carried out rapidly, efficiently to Different Individual II class MHC allelotrope, and then the gene of wherein banding pattern Different Individual is checked order, both avoid the waste that large scale sequencing brings, and also can shorten experimental period.
Accompanying drawing explanation
Fig. 1 is that primer Beta1085E2F/Beta1085E2R of the present invention carries out the electrophoresis detection result of pcr amplification to seven different Chinese alligator individualities.Marking with reference to molecular weight is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp successively.
Fig. 2 carries out gained electrophoresis result after SSCP somatotype to the Beta1085E2F/Beta1085E2R of the individual mhc genes of 12 the different Chinese alligators site PCR primer that increases.
Embodiment
Of the present invention a pair for the Chinese alligator II class MHC (majorhistocompatibilitycomplex that increases, MHC) primer of gene order, the mhc gene fragment that its basis is checked order obtained for early stage by BAC, there is the feature of larger difference according to mhc gene intron, use PrimerPremier5 to design site-specific primer.Once designed multipair primer during the course, showed that primer of the present invention was for optimum through experimental result.
Apply primer amplification Chinese alligator mhc gene of the present invention and the concrete grammar carrying out analyzing is as follows:
1, the DNA of Chinese alligator blood sample to be measured is extracted by phenol-chloroform extraction process, as template;
2, the qualified DNA of extraction is carried out pcr amplification, 10 μ LPCR reaction systems are: Chinese alligator poba gene group DNA0.8 μ L, each 0.2 μ L of upstream and downstream primer, ultrapure water 4.4 μ L, 2 × UltraTaqMasterMix4.4 μ L.PCR response procedures is: 95 DEG C of denaturations 5 minutes, 95 DEG C of sex change 30 seconds, and 63 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, 34 circulations, and last 72 DEG C of extensions 10 minutes, product 4 DEG C saves backup.
3, carry out separation detection to amplified production, concrete operation step is as follows:
1) with glass washing composition cleaning size sheet glass, with alcohol swab wiping 3 times after draining, and treat alcohol volatilization post-erection plate, spacer bar is placed between size sheet glass, and with special frame, both sides is clamped, be fixed on mould;
2) prepare the non-denaturing polyacrylamide gel glue of 12%, slowly inject between size sheet glass after glue is mixed, confirm that bubble-free inserts comb, normal temperature horizontal after existing;
3) after gelling is solid, unload also frame from mould and, on electrophoresis apparatus, use agarose sealing; Take off comb, and with filling the irrigation with syringe loading wells of 0.5 × TBE; Put into electrophoresis chamber, pour 0.5 × TBE into, prerunning 20min;
4) sample electrophoresis, draws 7uLPCR product, adds 4uL2 × loadingbuffer, quick centrifugal mixing, be placed on ice rapidly, and add in time in loading wells with rifle, 4 DEG C, 35W, electrophoresis 6-7h after 95 DEG C of sex change 4min30s;
5) glue is unloaded between sheet glass, add 500mL stationary liquid, on shaking table, fix 30min, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
6) add 500mL silver dye liquor, on shaking table, silver dye 30min, outwells silver-colored dye liquor, uses 500mL distilled water to wash rapidly glue 4 times;
7) add the developing solution of 500mL precooling, in shaking table development, wait for that band removes developing solution after clear, add 500mL stationary liquid and stop development, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
8) read band, determine genotype.
Below in conjunction with example, invention is described further:
The individual mhc gene of the different Chinese alligator of Beta1085E2F/Beta1085E2R primer pair is utilized to increase:
1.DNA extracts: from-20 DEG C of refrigerators, take out the Chinese alligator blood sample being added with EDTA anti-freezing, thaw, get 20 μ L in 1.5mL centrifuge tube, after digestion is spent the night, extracts the genomic dna in blood sample with phenol-chloroform extraction process.By the concentration of spectrophotometer Detection and Extraction gained DNA solution, length range and the quality of DL2000DNAmarker Detection and Extraction gained DNA fragmentation is coordinated with 1% agarose gel electrophoresis, get a little DNA stoste and be diluted to the diluent that concentration is about 200ng/ μ L, preserve under-20 DEG C of conditions.
2.PCR increases: with qualified DNA diluent for template, the Beta1085E2F/Beta1085E2R primer utilizing the present invention to design carries out pcr amplification.PCR amplification system is 10 μ L: Chinese alligator genomic dna diluent 0.8 μ L, each 0.2 μ L of upstream and downstream primer, ultrapure water 4.4 μ L, 2 × UltraTaqMasterMix4.4 μ L.PCR response procedures is: 95 DEG C of denaturations 5 minutes, 95 DEG C of sex change 30 seconds, and 63 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 34 circulations, last 72 DEG C of extensions 10 minutes.Coordinate DL2000DNAmarker to detect the quality of amplified production with 1% agarose gel electrophoresis, result shows, and this all to increase smoothly the single band obtaining becoming clear to primer in Chinese alligator DNA sample, presents stable site-specific and to increase ability.
3. pair PCR primer carries out SSCP somatotype, and step is as follows:
1) with glass washing composition cleaning size sheet glass, with alcohol swab wiping 3 times after draining, and treat alcohol volatilization post-erection plate, spacer bar is placed between size sheet glass, and with special frame, both sides is clamped, be fixed on mould;
2) prepare the non-denaturing polyacrylamide gel glue of 12%, slowly inject between size sheet glass after glue is mixed, confirm that bubble-free inserts comb, normal temperature horizontal after existing;
3) after gelling is solid, unload also frame from mould and, on electrophoresis apparatus, use agarose sealing; Take off comb, and with filling the irrigation with syringe loading wells of 0.5 × TBE; Put into electrophoresis chamber, pour 0.5 × TBE into, prerunning 20min under 35W power;
4) sample electrophoresis, draws 7uLPCR product, adds 4uL2 × loadingbuffer, quick centrifugal mixing, be placed on ice rapidly, and add in time in loading wells with rifle, 4 DEG C, 35W, electrophoresis 6h30min after 95 DEG C of sex change 4min30s;
5) glue is unloaded between sheet glass, add 500mL stationary liquid, on shaking table, fix 30min, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
6) add 500mL silver dye liquor, on shaking table, silver dye 30min, outwells silver-colored dye liquor, uses 500mL distilled water to wash rapidly glue 4 times;
7) add the developing solution of 500mL precooling, in shaking table development, wait for that band removes developing solution after clear, add 500mL stationary liquid and stop development, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
8) read band, determine genotype.
4. SSCP genotyping result such as the Fig. 2 read shows, and Chinese alligator mhc gene demonstrates three kinds of different banding patterns on Beta1085E2F/Beta1085E2R increases site, can be divided into AA type, BB type and AB type according to banding pattern.
Claims (6)
1. the unit point specificity amplification primer of II class mhc gene detected for Chinese alligator antibacterium potentiality for a pair, under the title of primer and concrete sequence:
Upstream primer: Beta1085E2F:CTCTGCCCAGTAGGAGCCGTGC;
Downstream primer: Beta1085E2R:AACAAAGACCCCAGGAATCCAG.
2. the classifying method of II class mhc gene of a Chinese alligator antibacterium potentiality detection, it is characterized in that adopting PCR primer pair described in claim 1, carry out PCR reaction, often pair of primer all carries out target fragment amplification in 10 μ LPCR systems, is then utilized by amplified production SSCP to carry out gene type respectively.
3. method according to claim 2, is characterized in that 10 described μ LPCR systems are: Chinese alligator genes of individuals group DNA0.8 μ L, each 0.2 μ L of upstream and downstream primer, ultrapure water 4.4 μ L, 2 × UltraTaqMasterMix4.4 μ L.
4. method according to claim 2, is characterized in that, the program of described PCR reaction is: 95 DEG C of denaturations 5 minutes, 95 DEG C of sex change 30 seconds, and 63 DEG C of annealing 30 seconds, 72 DEG C extend 30 seconds, totally 34 circulations, last 72 DEG C of extensions 10 minutes.
5. method according to claim 2, is characterized in that in described SSCP classifying method, distilled water should be used respectively to wash glue 4 times, to obtain SSCP band clearly after fixing, after silver dye and after colour developing.
6. method according to claim 2, is characterized in that the method for described SSCP somatotype is:
1) with glass washing composition cleaning size sheet glass, with alcohol swab wiping 3 times after draining, and treat alcohol volatilization post-erection plate, spacer bar is placed between size sheet glass, and with special frame, both sides is clamped, be fixed on mould;
2) prepare the non-denaturing polyacrylamide gel glue of 12%, slowly inject between size sheet glass after glue is mixed, confirm that bubble-free inserts comb, normal temperature horizontal after existing;
3) after gelling is solid, unload also frame from mould and, on electrophoresis apparatus, use agarose sealing; Take off comb, and with filling the irrigation with syringe loading wells of 0.5 × TBE; Put into electrophoresis chamber, pour 0.5 × TBE into, prerunning 20min;
4) sample electrophoresis, draws 7uLPCR product, adds 4uL2 × loadingbuffer, quick centrifugal mixing, be placed on ice rapidly, and add in time in loading wells with rifle, 4 DEG C, 35W, electrophoresis 6-7h after 95 DEG C of sex change 4min30s;
5) glue is unloaded between sheet glass, add 500mL stationary liquid, on shaking table, fix 30min, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
6) add 500mL silver dye liquor, on shaking table, silver dye 30min, outwells silver-colored dye liquor, uses 500mL distilled water to wash rapidly glue 4 times;
7) add the developing solution of 500mL precooling, in shaking table development, wait for that band removes developing solution after clear, add 500mL stationary liquid and stop development, outwell stationary liquid, pour 500mL distilled water into and wash glue 4 times;
8) read band, determine genotype.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588240A (en) * | 2018-06-05 | 2018-09-28 | 浙江大学 | Chinese alligator microsatellite polymorphism site, identification method and specific primer sequences |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588240A (en) * | 2018-06-05 | 2018-09-28 | 浙江大学 | Chinese alligator microsatellite polymorphism site, identification method and specific primer sequences |
| CN108588240B (en) * | 2018-06-05 | 2019-09-13 | 浙江大学 | Chinese alligator microsatellite polymorphism site, identification method and specific primer sequences |
| CN112813171A (en) * | 2020-12-17 | 2021-05-18 | 水利部中国科学院水工程生态研究所 | MHC gene primer for cupreous rotundifolia fish and application thereof |
| CN112813171B (en) * | 2020-12-17 | 2023-05-26 | 水利部中国科学院水工程生态研究所 | MHC gene primer for round-mouth copper fish and application thereof |
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