CN102604897A - Application of lentivirus containing carp IGF2b gene in increase of carp meat yield - Google Patents
Application of lentivirus containing carp IGF2b gene in increase of carp meat yield Download PDFInfo
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- CN102604897A CN102604897A CN2012100876219A CN201210087621A CN102604897A CN 102604897 A CN102604897 A CN 102604897A CN 2012100876219 A CN2012100876219 A CN 2012100876219A CN 201210087621 A CN201210087621 A CN 201210087621A CN 102604897 A CN102604897 A CN 102604897A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention relates to application of a lentivirus containing a carp IGF2b gene in increase of carp meat yield. According to the invention, non-mitotic cells and mitotic cells in a specific tissue of a carp are infected with the lentivirus containing the carp IGF2b gene, and the expression of a target gene in the specific tissue is affected, so that the growth status of the carp is changed. A result shows that when carp larvae are infected with a lentivirus that contains the carp IGF2b gene according to a detection result, after three months, compared with a control group, the weight of the carps in an infection group is significantly increased, in addition, each index of carp blood is not affected, and the safety is good.
Description
Technical field
The invention belongs to molecular biology fish breeding technical field, relate in particular to a kind of slow virus that contains carp IGF2b gene in the application that improves on the carp meat yield.
Background technology
At present; The carp that obtains faster growing speed still is the demand of industry development; Therefore, select fast growth, colory carp kind; Especially select good candidate gene and proper process method when improving carp growth speed, not to influence the expression of other proterties, have great importance.
The method that microinjection, sperm vector method, liposome transfection method, retrovirus vector method, nuclear transfer method etc. improve the animal varieties performance in traditional protokaryon is all because of cost is high, efficient is hanged down or inserted shortcomings such as fragment is little can't widespread use.
The slow virus that contains carp IGF2b gene is a kind of viral liquid that obtains with the method for artificial separation and modification; Promptly reverse transcription is cDNA according to carp RNA; Design corresponding primer again and carry out pcr amplification; Obtain to contain the gene fragment of IGF2b with corresponding restriction endonuclease cutting, obtain to contain the slow virus of carp IGF2b gene thereby finally use this gene fragment to be connected with carrier pLenti6.3-IRES-EGFP; Its concrete preparation method sees that number of patent application is described in 201110356210.0 the patent application document.
Handle carp with this slow virus that contains carp IGF2b gene and infect non-division stage and division stage cell in the carp particular organization, thus influence particular organization's target gene expression consequently its upgrowth situation change.This kind breeding method is applied to fish can confirm to have the strain of goal gene in a short time; Improve the carp meat yield; Thereby shorten the fish breeding time, and can make and be integrated in long-term, the stable expression of the genomic goal gene of target cell, this type of research does not appear in the newspapers so far.
Summary of the invention
The technical problem that solves: the objective of the invention is to overcome the deficiency that exists in the prior art, provide a kind of slow virus that contains carp IGF2b gene in the application that improves on the carp meat yield.
Technical scheme:
Concrete treatment step is following:
(1) obtains to contain the slow virus of carp IGF2b gene with the method for artificial separation and modification;
(2) let carp infect said slow virus, i.e. the said slow virus of injection in the carp body;
(3) confirm with green fluorescence whether infection is successful: after infecting 48h, the expression situation through the fluorescent microscope observation method is observed green fluorescent protein if any the expression of green fluorescent protein, shows that then said carp has infectd said slow virus;
As further improvement of the present invention, the infection of slow virus described in the step (2) was accomplished in the juvenile fish stage.
Further improve as of the present invention, in the step (2), inject said slow virus during for 5g in said carp body weight.
As a kind of preferred version of the present invention, the carp injection site is a muscle of back in the step (2).
As further preferred version of the present invention, the carp injection site is fix a point the foremost intermediate point of side line vertical range of dorsal fin in the step (2).
The present invention comprises that also observation causes the step (4) of the variation of carp proterties owing to infection: after 3 months, and checking carp growth situation and physiochemical indice situation.
Beneficial effect
After 3 months, upgrowth situation and the physiochemical indice situation of carp infected in checking, and its verification method is according to negative control, positive control, contrasts changes of weight situation and the changing condition of physiochemical indice of the carp of different IDs.The result shows that with the slow virus infection carp young that contains carp IGF2b gene that has detected, infected group is compared control group, and the carp body weight has obtained the effect of remarkable increase, and dosage better effects if when big, and to the not influence of carp physiochemical indice, security is good simultaneously.
This kind breeding method is applied to fish can confirm to have the strain of goal gene in a short time; Shorten the fish breeding time; And can make to be integrated in long-term, the stable expression of the genomic goal gene of target cell, owing to physiochemical indice is not impacted, so security is good.
Description of drawings
Fig. 1 can show by the growth result of the carp muscle of back of slow virus infection have the green fluorescence explicit declaration to be infected for fluorescent microscope detects the expression of fish body green fluorescent protein behind the slow virus injection 48h through this expression
Fig. 2 cultures the difference of the weight character of different experiments group fish after 3 months
Embodiment
Embodiment 1
For the control test error, all injection work are accomplished by a people, and all experimental group are all cultured in the same pool, and all aquaculture managements are undertaken by a people.
(1) slow virus that contains carp IGF2b gene is from the compound breeding of China Aquatic Science Research Academy Fresh Water Fishery Research Center carp seminar; Its preparation method is 201110356210.0 patent application document referring to number of patent application; 250 tail carps are taken from Nan Quan cultivation base, fresh water fishery research centre, China Aquatic Science Research Institute Wuxi, set up 5 experimental group altogether:
One is the negative control group that is left intact;
One is the positive controls of injecting the slow virus diluent that does not contain carp IGF2b gene, and injection volume is 20 μ L, amounts to contain 1.1 * 10 approximately
5Individual virus;
Other three is the slow virus infection group that contains carp IGF2b gene slow virus diluent according to basic, normal, high three different concns gradients injection, and its ID is respectively 10 μ L, 20 μ L and 40 μ L, and to contain viral number be 5.54 * 10 to correspondence respectively
4Individual virus, 1.1 * 10
5Individual virus, 2.22 * 10
5Individual virus.
(2) behind the 48h, each 2 tail of picked at random negative control group, positive controls and each dosage infected group fish carry out green fluorescent protein (GFP) fluoroscopic examination.The result shows that each dosage infected group fish has all infected carp IGF2b gene slow virus, sees Fig. 1;
(3) on the basis of (2), culture after 3 months, measure body weight, calculate the growth performance between the different experiments group fish.The result shows that the carp that infects carp IGF2b gene slow virus is obviously bigger than not infecting this viral carp body weight, and the body weight of the highest infective dose 40uL group is maximum, sees Fig. 2.
(4) on the basis of (3), measured the physiochemical indice of different experiments group fish, comprise albumen totally 9 indexs on kidney, liver and the IGF2 signal transduction path, measuring sample size is 20 tail fishes, the result sees the following form.
Annotate: different letter marks are represented significant difference, do not have the letter mark or have same letter to represent difference not remarkable
Show by last table: all do not have significant difference between 8 index negative control group such as SEAP, total protein, blood urea nitrogen, creatinine, total cholesterol, rhIGF-1 2, IGF-1 1, IGF-1 2 and the positive controls, have only this index positive controls of alanine transaminase to be higher than negative control group;
Once more, creatinine, rhIGF-1 2, IGF-1 1 and IGF-1 2 do not detect significant difference yet between infected group and control group;
At last, 10 μ L virus liquid infected group makes alanine transaminase in the blood, SEAP, blood urea nitrogen and total protein concentration increase, and 20 μ L virus liquid infected group and 40 μ L virus liquid infected group total cholesterol concentration reduce.
Comprehensive above step, we can obtain, and carry out the injection of carp muscle of back with the slow virus that contains carp IGF2b gene; The IGF2b animal model of expression can be obtained to cross,, the infectious effect of slow virus can be confirmed according to fluoroscopic examination at the carp back; According to the detection of growth differences between different experiments group after 3 months, can confirm the influence degree of carp IGF2b gene overexpression to the carp changes of weight: the result shows, with the slow virus infection carp young that contains carp IGF2b gene that has detected; Infected group is compared control group, and the carp body weight has obtained the effect of remarkable increase, and dosage better effects if when big; To the not influence of carp physiochemical indice, security is good simultaneously.
Embodiment 2
Different is with embodiment 1, and the carp injection site is fix a point the foremost intermediate point of side line vertical range of dorsal fin in the step (2).
Its beneficial effect is for can guarantee more that the infected degree of carp is identical on the same group.
Claims (6)
1. a slow virus that contains carp IGF2b gene is realized in the application that improves on the carp meat yield as follows:
(1) obtains to contain the slow virus of carp IGF2b gene with the method for artificial separation and modification;
(2) carp infects said slow virus: the said slow virus of injection in the carp body;
(3) confirm to infect whether success with green fluorescence: after infecting 48h,, successfully infect the expression that green fluorescent protein is arranged in the carp body of slow virus through the expression situation that the fluorescent microscope observation method is observed green fluorescent protein.
2. the slow virus that contains carp IGF2b gene according to claim 1 is in the application that improves on the carp meat yield, and it is characterized by: the infection of slow virus described in the step (2) was accomplished in the juvenile fish stage.
3. the slow virus that contains carp IGF2b gene according to claim 1 is characterized by in the application that improves on the carp meat yield: in the step (2), inject said slow virus during for 5g in the carp body weight.
4. according to each described slow virus that contains carp IGF2b gene is in the application that improves on the carp meat yield in the claim 1～3, it is characterized by: the carp injection site is a muscle of back in the step (2).
5. it is characterized by in the application that improves on the carp meat yield according to each described slow virus that contains carp IGF2b gene in the claim 1～3: the carp injection site is fix a point the foremost intermediate point of side line vertical range of dorsal fin in the step (2).
6. according to each described slow virus application on raising carp meat yield that contains carp IGF2b gene in the claim 1～3; It is characterized by: also comprise step (4): observation is owing to infecting the variation that causes the carp proterties: infect said slow virus after 3 months carp, upgrowth situation and the physiochemical indice situation of checking carp.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555791A (en) * | 2013-10-28 | 2014-02-05 | 中国水产科学研究院淡水渔业研究中心 | Clustering method for inspecting influence of slow viruses containing carp IGF2b (Insulin Growth Factor 2b) gene on physiques of carps |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1487083A (en) * | 2003-07-10 | 2004-04-07 | 中山大学 | Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof |
| CN102250912A (en) * | 2011-06-10 | 2011-11-23 | 中国水产科学研究院淡水渔业研究中心 | Construction method for lentiviral vector containing IGF2b gene |
| CN102363792A (en) * | 2011-11-11 | 2012-02-29 | 中国水产科学研究院淡水渔业研究中心 | Lentivirus method for preparing IGF2b transgenic fish |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1487083A (en) * | 2003-07-10 | 2004-04-07 | 中山大学 | Grouper insulin-like growth factor II gene, carrier and recombinant strain containing the gene and application thereof |
| CN102250912A (en) * | 2011-06-10 | 2011-11-23 | 中国水产科学研究院淡水渔业研究中心 | Construction method for lentiviral vector containing IGF2b gene |
| CN102363792A (en) * | 2011-11-11 | 2012-02-29 | 中国水产科学研究院淡水渔业研究中心 | Lentivirus method for preparing IGF2b transgenic fish |
Non-Patent Citations (4)
| Title |
|---|
| JYH-YIH CHEN ET AL: "Expression of recombinant tilapia insulin-like growth factor-I and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides", 《AQUACULTURE》, vol. 181, no. 34, 15 January 2000 (2000-01-15), pages 347 - 360 * |
| SHUMING ZOU ET AL: "Zebrafish IGF genes: gene duplication, conservation and divergence, and novel roles in midlinge and notochord development", 《PLOS ONE》, vol. 4, no. 9, 17 September 2009 (2009-09-17), pages 7026 * |
| TSE MC ET AL: "PCR-cloning and gene expression studies in common carp (cyprinus carpio) insulin-like growth factor-II", 《BIOCHIMICA ET BIOPHYSICA ACTA》, vol. 1575, no. 13, 3 May 2002 (2002-05-03), pages 63 - 74 * |
| YVONNE A R WHITE ET AL: "Targeted gene knockdown in Zebrafish reveals distinct intraembryonic functions for insulin-like growth factor II signaling", 《ENDOCRINOLOGY》, vol. 150, no. 9, 14 May 2009 (2009-05-14), pages 4366 - 4375 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555791A (en) * | 2013-10-28 | 2014-02-05 | 中国水产科学研究院淡水渔业研究中心 | Clustering method for inspecting influence of slow viruses containing carp IGF2b (Insulin Growth Factor 2b) gene on physiques of carps |
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| CN102604897B (en) | 2014-04-16 |
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