CN104357559A - Identification method for key ribosomal proteins in developmental regulation of female schistosoma japonicum katsurada - Google Patents
Identification method for key ribosomal proteins in developmental regulation of female schistosoma japonicum katsurada Download PDFInfo
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Abstract
The invention relates to an identification method for key ribosomal proteins in developmental regulation of female schistosoma japonicum katsurada. The identification method comprises the following steps: using schistosoma japonicum cerariae to respectively infect mice; breeding the infected mice in an SPF (Specific Pathogen Free) level animal house; respectively collecting monosexually and bisexually infected female schistosoma japonicum katsurada; respectively carrying out solexa analysis and iTRAQ analysis on the female schistosoma japonicum katsurada; contrasting the ribosomal protein expression profile and gene expression profile in four developmental stages to obtain the expressional characteristics of the ribosomal proteins in the two levels of proteins and genes in different developmental stages of the female schistosoma japonicum katsurada. Compared with the prior art, through the Solexa and iTRAQ combinatory analysis of the ribosomal gene expression profile and the protein expression profile of the female schistosoma japonicum katsurada before and after copulation of female and male schistosoma japonicum katsurada and in combination with real-time PCR and western technical identification, the key ribosomal proteins in the developmental regulation of female schistosoma japonicum katsurada can be effectively determined. The identification of proteins provides a key basis for the research and development of the control measures of interfering the development of the schistosoma japonicum katsurada in a host and maintaining the concomitant immune protection force of the host for the purpose of influencing the ribosomal function of the schistosoma japonicum katsurada.
Description
Technical field
The invention belongs to life field of medical science, especially relate to the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation.
Background technology
After schistosomicide human body, the generation of a large amount of worm's ovums that sexual maturity causes and release are the basic reasons causing schistosomicide.In addition, after existing cured substance schistosomicide, patient still can because of Human Water Contact subinfection again, not there is immune protective efficiency.Research shows, the worm that lives can stimulate human body to produce the immune protective efficiency resisting again subinfection in human body, but the female worm itself that lives can be caused a disease because sexual maturity is laid eggs.Therefore, be badly in need of setting up a kind of method at present and schistosomicide in the infected's body can be regulated to grow, even if the infected keeps immunizing power for a long time, but be unlikely ill.Research finds, albumen synthetic cell device is not only by rrna, is again protein expression regulation and control person simultaneously.Especially, in schistosomicide sexual maturity growth course, personalized rrna is assemblied in during female worm sexual maturity is grown and plays a significant role.The crucial ribosomal protein of analysis and identification lays the foundation for the new prophylactico-therapeutic measures of research and development.
Summary of the invention
Object of the present invention is exactly the authentication method providing the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation in order to overcome defect that above-mentioned prior art exists, analyze female male schistosomiais by Solexa and iTRAQ to fill the span of a man's arms front and back gene expression profile and protein expression profiles, and in conjunction with real-time PCR and western technical identification, crucial ribosomal protein in female worm developmental regulation effectively can be determined.Identifying of this albumen will for by affecting for the purpose of polypide rrna function, to grow and the research and development maintaining the prophylactico-therapeutic measures of host's concomitant immunity protection provide crucial foundation to intervene polypide in host.
Object of the present invention can be achieved through the following technical solutions:
An authentication method for the crucial ribosomal protein of female Schistosoma japonicum worm developmental regulation, comprises the following steps:
(1) infection by cercariae of schistosoma mouse is used: comprise single spiral shell release Single Sex Cercariae and carry out unisexuality and infect discharge with many spiral shells and mix pair sexuality that cercaria carries out and contaminate two kinds of modes;
(2) raise at SPF level Animal House metainfective mouse, raise and comprise four periods of two stages, namely unisexuality infects 18 days and 23 days and two sexuality and contaminates 18 days and 23 days;
(3) Schistosoma japonicum in rear Mice Body is cultivated in collection: if single mouse only obtains female worm, as the female worm sample of unisexuality originally, if single mouse obtains both sexes polypide, is separated female worm, as the female worm sample of two property originally;
(4) the female worm of step (3) gained is transferred in Trizol reagent, be solexa and analyze: analyze different female this gene expression profile of worm sample respectively, analysis and comparison different sample Ribosomal Gene Expres difference;
(5) the female worm of step (3) gained is transferred in physiological saline, be iTRAQ and analyze: analyze different female this ribosomal protein of worm sample express spectra respectively, analysis and comparison different sample ribosomal protein differential expression;
(6) by contrast different developmental phases ribosomal protein express spectra and gene expression profile, obtain female worm in different developmental phases, ribosomal protein is at the expression characteristic of albumen and gene two levels.
After step (4) does solexa analysis, by Real-time PCR method checking solexa technical Analysis quality.
Be after iTRAQ analyzes in step (5), Western blot checking is carried out to the albumen of wherein high expression level.
When infecting mouse in step (1), utilize single spiral shell to discharge the feature of Single Sex Cercariae, by miracidium infection mouse of a spiral shell release, or combine postoperative infection mouse with the schistosomulum that different spiral shell discharges.
To metainfective mouse when SPF level Animal House raises 18 days, schistosomicide is rudimentary state before filling the span of a man's arms, and when being cultured to 23 days, be sexually matured state after filling the span of a man's arms, the mouse of getting these two time points is analyzed.
The method of collecting the Schistosoma japonicum after cultivating in Mice Body is: pour into from mouse aorta with aseptic pre-cold saline, go out schistosomicide, then schistosomicide is washed 3 times in stroke-physiological saline solution.
Be before solexa analyzes, under anatomical lens, with syringe needle picking and the female worm of transfer fresh in the EPP pipe containing 1000ul Trizol reagent ,-80 DEG C save backup, or RNA is got in direct homogenate.
Be iTRAQ analyze before, under anatomical lens, with syringe needle picking and the female worm of transfer fresh in the EPP pipe containing 1000ul physiological saline, save backup in liquid nitrogen.
Because the female worm of different developmental phases can express different ribosomal protein, assembling forms different rrna, i.e. personalized rrna, to complete specific demand and the function of different developmental phases.Analyze based on this, can obtain etap specific expressed ribosomal protein, this be choose the further investigation that different ribosomal protein carries out being intended to intervention rrna function pointedly to provide condition.Therefore, rrna assembling, polypide etap and Stage-specific protein are expressed and are connected by the foundation of the inventive method, analyze female male schistosomiais by Solexa and iTRAQ to fill the span of a man's arms front and back gene expression profile and protein expression profiles, and in conjunction with real-time PCR and western technical identification, crucial ribosomal protein in female worm developmental regulation effectively can be determined.Identifying of this albumen will for by affecting for the purpose of polypide rrna function, to grow and the research and development maintaining the prophylactico-therapeutic measures of host's concomitant immunity protection provide crucial foundation, for the similar investigation and application of close species provides foundation to intervene polypide in host.
Accompanying drawing explanation
Fig. 1 is that Schistosoma japonicum unisexuality and two sexy Ribosomal Gene Expres spectrum when educating 18 days (not growing before filling the span of a man's arms) and 23 days (after filling the span of a man's arms sexual maturity) of having hair dyed compare;
Gene differential expression situation in rrna metabolic pathway: A.23SSI (unisexuality infects 23 days) and 18SSI (unisexuality infects 18 days); B.18SSI with 18DSI (two sexuality dye 18 days); C.23DSI (two sexuality dye 23 days) and 18DSI; D.23DSI with 23SSI. upward arrow: gene upregulation is expressed; Lower arrow: gene deregulation is expressed.
E.18SSI and 18SSI Solexa analyzes ribosomal protein differential expression situation in different sample:; And 23SSI F.18SSI; And 23DSI G.23SSI; And 23DSI H.18DSI;
Fig. 2 is Realtime pcr analysis three ribosomal protein (CAX72575.1, CAX77721.1, CAX78924.1) at Schistosoma japonicum unisexuality and two sexy differential expression of having hair dyed when educating 18 days (not growing before filling the span of a man's arms) and 23 days (after filling the span of a man's arms sexual maturity);
Fig. 3 is the western blot analytical results of ribosomal protein RPL8 and RPL11 when growing 18 days and 23 days and unisexuality infection growth 23 days after the two sexy dye of Schistosoma japonicum.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
Embodiment
With Solexa technical Analysis gene expression profile, under method is shown in:
(1) utilize single spiral shell to discharge the feature of Single Sex Cercariae, by miracidium infection mouse of a spiral shell release, obtain the principle of parthenita.
(2) 50 mouse are infected respectively with at least 50 spiral shells, then, the cercaria combination discharged with different spiral shell, then infect at least 20 mouse.18 days and 23 days are raised at SPF level Animal House.If single mouse only obtains female worm, can be used as the female worm sample of unisexuality originally; If single mouse obtains both sexes polypide, be separated female worm, as the female worm sample of two property originally.
(3) female worm is collected respectively at the 18th day and 23 days.First pour into from mouse aorta with aseptic pre-cold saline, go out 18 days or 23 days schistosomicide, in stroke-physiological saline solution, wash 3 times.
(4) under anatomical lens, with syringe needle picking and the female worm of transfer fresh: transfer in the EPP pipe containing 1000ulTrizol reagent ,-80 degree save backup, or directly RNA (method is shown in Trizol working instructions) is got in homogenate.Be solexa and analyze use.Hua Da genome company has assisted sample gene expression spectrum analysis.
Different female this gene expression profile of worm sample is analyzed respectively, analysis and comparison different sample Ribosomal Gene Expres difference by solexa technology.See Fig. 1 (E, F, G and H).Gene expression profile after Solexa analyzes discloses, and after filling the span of a man's arms, female worm ribosomal protein gene differential expression feature is obvious.Prompting, the female worm after filling the span of a man's arms may assemble personalized rrna based on ribosomal protein differential expression.
Meanwhile, by Real-time PCR method checking solexa technical Analysis quality.Wherein, with following three ribosomal protein genes for standard (see table 1), as the Quality Control that Solexa analyzes.
Table 1. ribosomal protein gene and primer sequence
Solexa is female worm ribosomal protein gene differential expression spectrum before and after analyzing and obtaining filling the span of a man's arms, for further in protein level analysis with determine that differential expression ribosomal protein still needs protein expression spectrum analysis.
With iTRAQ technical Analysis protein expression profiles, under method is shown in:
(1) utilize single spiral shell to discharge the feature of Single Sex Cercariae, by miracidium infection mouse of a spiral shell release, obtain the principle of parthenita.
(2) 50 mouse are infected respectively with at least 50 spiral shells, then, the cercaria combination discharged with different spiral shell, then infect at least 20 mouse.18 days and 23 days are raised at SPF level Animal House.
(3) female worm is collected respectively at the 18th day and 23 days.First pour into from mouse aorta with aseptic pre-cold saline, go out 18 days or 23 days schistosomicide, in stroke-physiological saline solution, wash 3 times.If single mouse only obtains female worm, can be used as the female worm sample of unisexuality originally; If single mouse obtains both sexes polypide, be separated female worm, as the female worm sample of two property originally.
(4) under anatomical lens, with syringe needle picking and the female worm of transfer fresh: transfer in the EPP pipe containing 1000ul physiological saline, save backup in liquid nitrogen.Be iTRAQ and analyze use, sample protein expression spectrum analysis has been assisted in protein groups research centre, Beijing.
Different female this ribosomal protein of worm sample express spectra is analyzed respectively, analysis and comparison different sample ribosomal protein differential expression by iTRAQ technology.In Table 2-4.
Table 2. unisexuality infects 23 days (23SSI) and contaminates comparing of iTRAQ and the Solexa analytical results that 23 days (23DSI) female worm difference ribosomal proteins express with two sexy
The sexy dye 23 days (23DSI) of table 3. pair and two sexuality contaminate comparing of iTRAQ and the Solexa analytical results that 18 large (18DSI) female worm difference ribosomal proteins express
Table 4. unisexuality infects 23 days (23SSI) and unisexuality and infects comparing of iTRAQ and the Solexa analytical results that 18 days (18SSI) female worm difference ribosomal proteins express
By contrast different developmental phases ribosomal protein express spectra and gene expression profile, can the female worm of clear announcement different developmental phases (before filling the span of a man's arms and after filling the span of a man's arms), ribosomal protein is at the expression characteristic of albumen and gene two levels.
For verifying the reliability that proteinogram is analyzed further, to the albumen of wherein high expression level, as 60S ribosomalprotein L8 and Ribosomal protein L11 carries out Western blot checking, see Fig. 2.Result shows, in the proteinogram obtained by iTRAQ method, high expression level protein 60 S ribosomal protein L8 and Ribosomalprotein L11 also presents high expression level in Western blot analytical results, the reliability of proteinogram analytical procedure in analyzing to point out this.
In a word, above analysis is clear to be shown, the female worm of different developmental phases can express different ribosomal protein, and assembling forms different rrna, i.e. personalized rrna, to complete specific demand and the function of different developmental phases.Analyze based on this, can obtain etap specific expressed ribosomal protein, this be choose the further investigation that different ribosomal protein carries out being intended to intervention rrna function pointedly to provide condition.Therefore, rrna assembling, polypide etap and Stage-specific protein are expressed and are connected by the foundation of the method, for important foundation has been established in the screening of Schistosoma japonicum new drug target and new generation vaccine research and development.Meanwhile, for the similar investigation and application of close species provides foundation.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (8)
1. an authentication method for the crucial ribosomal protein of female Schistosoma japonicum worm developmental regulation, is characterized in that, comprise the following steps:
(1) infection by cercariae of schistosoma mouse is used: the schistosomicide Single Sex Cercariae unisexuality of carrying out comprising the release of single spiral shell infects pair sexuality that cercaria carries out that mixes discharge with many spiral shells and contaminates two kinds of modes;
(2) raise at SPF level Animal House metainfective mouse, raise and comprise four periods of two stages, namely unisexuality infects 18 days and 23 days and two sexuality and contaminates 18 days and 23 days;
(3) Schistosoma japonicum in rear Mice Body is cultivated in collection: if single mouse only obtains female worm, as the female worm sample of unisexuality originally, if single mouse obtains both sexes polypide, is separated female worm, as the female worm sample of two property originally;
(4) the female worm of step (3) gained is transferred in Trizol reagent, be solexa and analyze: analyze different female this gene expression profile of worm sample respectively, analysis and comparison different sample Ribosomal Gene Expres difference;
(5) the female worm of step (3) gained is kept in physiological saline, is iTRAQ and analyzes: analyze different female this ribosomal protein of worm sample express spectra respectively, analysis and comparison different sample ribosomal protein differential expression;
(6) by contrast different developmental phases ribosomal protein express spectra and gene expression profile, obtain female worm in different developmental phases, ribosomal protein is at the expression characteristic of albumen and gene two levels.
2. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, is characterized in that, after step (4) does solexa analysis, by Real-time PCR method checking solexa technical Analysis quality.
3. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, is characterized in that, is after iTRAQ analyzes in step (5), carries out Western blot checking to the albumen of wherein high expression level.
4. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, it is characterized in that, when mouse being infected in step (1), single spiral shell is utilized to discharge the feature of Single Sex Cercariae, by miracidium infection mouse of a spiral shell release, or combine postoperative infection mouse with the cercaria that different spiral shell discharges.
5. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, it is characterized in that, to metainfective mouse when SPF level Animal House raises 18 days, schistosomicide is rudimentary state before filling the span of a man's arms, when being cultured to 23 days, for sexually matured state after filling the span of a man's arms, the mouse of getting these two time points is analyzed.
6. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, it is characterized in that, the method of collecting the Schistosoma japonicum after cultivating in Mice Body is: pour into from mouse aorta with aseptic pre-cold saline, go out schistosomicide, then schistosomicide is washed 3 times in stroke-physiological saline solution.
7. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, it is characterized in that, before doing solexa analysis, under anatomical lens, with syringe needle picking and the female worm of transfer fresh in the EPP pipe containing 1000ul Trizol reagent,-80 DEG C save backup, or directly RNA is got in homogenate.
8. the authentication method of the crucial ribosomal protein of a kind of female Schistosoma japonicum worm developmental regulation according to claim 1, it is characterized in that, before doing iTRAQ analysis, under anatomical lens, with syringe needle picking and the female worm of transfer fresh in the EPP pipe containing 1000ul physiological saline, save backup in liquid nitrogen.
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| CN115961013A (en) * | 2019-06-18 | 2023-04-14 | 中国医学科学院病原生物学研究所 | Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification |
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| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
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| CN103119179A (en) * | 2010-07-23 | 2013-05-22 | 哈佛大学校长及研究员协会 | Methods for detecting signatures of disease or conditions in bodily fluids |
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| JUN SUN: "Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing", 《PARASITOL RES》 * |
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| CN115961013A (en) * | 2019-06-18 | 2023-04-14 | 中国医学科学院病原生物学研究所 | Screening of schistosoma japonicum W chromosome specific gene and application thereof in cercaria sex identification |
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Application publication date: 20150218 |