35 results about "Head Kidney" patented technology

The invention provides a Chinese herbal medicine fodder additive for accelerating crucians to grow and enhancing the immunity and a fodder containing the additive. The Chinese herbal medicine fodder additive is formed by drying malts, hawthorns, green teas, lotus leaves, radix ophiopogonis and semen cassiae, and then performing crushing and uniform mixing. The Chinese herbal medicine fodder additive is added into a common crucian fodder so that the growing speed of the crucians can be obviously improved, the water content of muscles is reduced, the protein content of the muscles is improved and the fat content of the muscles is reduced; the hepatosomatic index can be reduced, and the spleen index and the head-kidney index can be improved; the blood serum T-SOD (Total-Superoxide Dismutase)can be obviously improved and the content of MDA (Malondialdehyde), TG (Triglyceride) and TC (cholesterol) is obviously reduced; the Chinese herbal medicine fodder additive has the characteristics ofgreenness, naturalness, no drug resistance, no residues, diversified functions, consideration of growth and disease resistance and the like; and the Chinese herbal medicine fodder additive can greatly reduce the breeding cost of the crucians and is suitable for industrial production.

The invention discloses a method for preparing chromosomes through large-scale barbel fish nephrocyte in-vivo culture and relates to a method for preparing fish chromosomes. The invention aims to solve the technical problem of the method for preparing the chromosomes of the large-scale barbel fish. The method comprises the following steps of: performing living body injection, namely injecting phytohaemagglutinin (PHA) into an abdominal cavity of the living body twice, and injecting colchicine; 2, sampling, namely washing head-kidney in NaCl normal saline, and collecting cells; 3, performing hypotension by using a mixed solution of sodium citrate and KCl; 4, performing cryofixation by using a Carnoy's fixing solution; 5, dropping through a cold drop method; and 6, staining through Giemsa staining solution, and obtaining the chromosomes of the large-scale barbel fish. The method is easy and convenient to operate and stable in result, and the large-scale barbel fish metacinesis specimen which is complete in number, good in dispersion and clear in shape can be obtained. The method is applied to the field of preparation of fish chromosomes.

Provided is technology of external model construction of pseudosciaena crocea head kidney macrophage. The fish head kidney macrophage is horizontally and centrifugally separated by utilizing Percoll with 31% / 45% gradient and is cultured in an L15 compound culture medium under the condition that the temperature is 22 DEG C. Each liter of the culture medium contains 50ml fetal calf serum, 0.1 million UI penicillin / streptomycin, 0.05 million UI heparin, 10mmol 14-ethoxyl-piperazine ethanesulfonic acid (HEPES) and 0.5g glucose. The pH of the culture medium is 7.4. The method and the application have the advantages that (1) the proportion of the pseudosciaena crocea head kidney macrophage which is centrifugally separated by utilizing Percoll with 31% / 45% gradient in the separation cell is as high as 42%; (2) the purity of the adherence pseudosciaena crocea head kidney macrophage after liquid culture is as high as 90%; (3) the livability of the pseudosciaena crocea head kidney macrophage after one week culture is higher than 58%; and (4) a function checking experiment proves that LPS can remarkably affect the swallow function and breath eruption activity of the cultured pseudosciaena crocea head kidney macrophage.

The invention discloses a method for improving the streptococcus iniae infection resisting capability of GIFT tilapia in high-density culture. The method comprises the steps of preparing of compound Chinese herbal medicine, adding, feeding and breeding management. Compared with the prior art, the method has the following advantages: (1) the immunological protection of the GIFT tilapia in high-density culture can be effectively improved, and the survival rate of GIFT tilapia infected with streptococcus iniae is improved; (2) the compound Chinese herbal medicine added in a feed belongs to environment-friendly feed additives, and has less negative effect on fish and water; (3) the blood and head-kidney immunological stress response can be effectively enhanced, and the liver stress injury is reduced; (4) the adopted technological steps of the method are simple, the Chinese herbal medicine is low, and easy to obtain and preserve, and can be well absorbed and utilized by a fish body, and excrement of the GIFT tilapia does not impact the water quality.

The invention discloses a preparation method of chromosomes of adult epinephelus akaara and belongs to the field of cell biology. The preparation method comprises the following steps: pretreatment of materials, preparation of kidney cell suspension, low-permeation treatment, fixation, dropping and dyeing, wherein the pretreatment of the materials comprises the steps of with an in vivo injection method of phytohemagglutinin (PHA), injecting PHA into the pectoral-fin base part of the experimental adult epinephelus akaara with 15 micrograms in each gram of the adult epinephelus akaara; after 24 hours, injecting colchicine solution with 2 micrograms in each gram of the adult epinephelus akaara; and after 2.5-3 hours, taking out the head kidney. The preparation method disclosed by the invention has the advantages that the method for one-time injection of PHA is adopted, simultaneously, the injection concentration is increased, and good effect can be obtained after 24 hours, so that the time is saved; and due to the characteristics of tissue cells of the epinephelus akaara, complete cells are not easily obtained when the low-permeation treatment is carried out in the prior art, so that the prior art is creatively changed and a clear chromosome image is obtained.

The invention discloses an establishment method of a cLYZ / hTLF double-antibacterial gene recombinant adenovirus, and a recombinant adenovirus and application thereof. The method comprises the following steps: respectively amplifying cLYZ gene and hTLF gene from grouper head-kidney tissues and human milk somatic cell total RNAs, cloning the obtained goal genes into a pDC315 shuttle vector, co-transfecting HEK293 cells through liposome by using the target-gene-containing shuttle plasmid and adenovirus framework plasmid, and packaging to obtain the high-titer recombinant adenovirus; and evaluating the combined inhibiting actions of the cLYZ gene and hTLF gene on the milk cow mastitis primary pathogens under the adenovirus mediated conditions.

The invention discloses a pseudosciaena crocea head kidney cell line and a construction method thereof, relating to cell lines. The pseudosciaena crocea head kidney cell line is a pseudosciaena crocea head kidney cell line LYCK with the preservation number of CCTCC NO: C2013113. The construction method of the pseudosciaena crocea head kidney cell line comprises the steps of (1) preparing a cell culture medium; (2) constructing primary culture; (3) performing secondary culture. The constructed pseudosciaena crocea head kidney cell line is fibrous, can be subjected to continuous passage, and is sub-cultured for 70 generations; a large quantity of pseudosciaena crocea head kidney cell lines can be provided; the pseudosciaena crocea head kidney cell line can be directly applied to research of various pseudosciaena crocea immunity related functional genes. The pseudosciaena crocea head kidney cell line can be applied to important researches including cytobiology, molecular biology, virology, toxicology, oncology, genetics, immunology and the like.

The invention relates to an epinephelus coioides interferon IFNgamma2 gene and a protein encoded by the sameas well as application of the protein in preparing a novel immunopotentiator. According to the invention, an open reading frame of the IFNgamma2 gene is cloned from epinephelus coioides head-kidney total RNA by specific primers SEQ ID NO:4 and SEQ ID NO:5 by means of a method of a primer amplification gene segment and RACE overall length amplification. The nucleotide sequence is shown as SEQ ID NO:2. The amino acid sequence of encoded protein is shown as SEQ ID NO:1. An initial test shows that the epinephelus coioides interferon IFNgamma2 protein can induce relevant immunogene expression and can be developed to natural fish immunopotentiator or immunologic adjuvant.

The invention relates to an epinephelus coioides interferon IFNgamma1 gene, a protein coded by the gene and an application of the protein in preparing a novel immunopotentiator. According to the invention, an opened reading frame of the IFNgamma1 gene is cloned from head-kidney RNAs (Ribonucleic Acid) of epinephelus coioides through a method of screening a genome database and designing specific primer amplification. The nucleotide sequence of the gene is shown as SEQIDNO:2. The amino acid sequence of the protein coded is shown as SEQIDNO:1. Through initial verification, the epinephelus coioides interferon IFNgamma1 protein can induce associated expression of immunogenes and can be developed to natural immunopotentiators or immunologic adjuvants for fishes.

The invention relates to a preparation method of a siganus guttatus chromosome. The preparation method comprises the following steps of: (1) taking out head-kidney tissues from siganus guttatus, placing in a culture dish containing physiological saline, cleaning, removing blood cots and other tissues, cutting the head-kidney tissues into pieces, and then fully homogenizing the head-kidney tissues to prepare a cell suspension; (2) performing hypotonicity and fixation; (3) performing fixed centrifugation, and then drilling on a slide; and (4) dyeing, drying the slide, then placing the slide under a microscope for observation, and selecting metaphase cells with good dispersion of the chromosome for performing photomicrometrology. The preparation method disclosed by the invention is simple and convenient in operation and stable in result, expensive reagents and special instruments are not required, and the metaphase mitosis in the siganus guttatus chromosome, which has the advantages of complete number, good dispersion, appropriate length, clear centromeres, appropriate separation of two monomers and clear shape, can be obtained by making the slide.

The invention provides a Chinese herbal medicine fodder additive for accelerating crucians to grow and enhancing the immunity and a fodder containing the additive. The Chinese herbal medicine fodder additive is formed by drying malts, hawthorns, green teas, lotus leaves, radix ophiopogonis and semen cassiae, and then performing crushing and uniform mixing. The Chinese herbal medicine fodder additive is added into a common crucian fodder so that the growing speed of the crucians can be obviously improved, the water content of muscles is reduced, the protein content of the muscles is improved and the fat content of the muscles is reduced; the hepatosomatic index can be reduced, and the spleen index and the head-kidney index can be improved; the blood serum T-SOD (Total-Superoxide Dismutase)can be obviously improved and the content of MDA (Malondialdehyde), TG (Triglyceride) and TC (cholesterol) is obviously reduced; the Chinese herbal medicine fodder additive has the characteristics ofgreenness, naturalness, no drug resistance, no residues, diversified functions, consideration of growth and disease resistance and the like; and the Chinese herbal medicine fodder additive can greatly reduce the breeding cost of the crucians and is suitable for industrial production.

The invention discloses an epinephelus lanceolatus head kidney cell line as well as a construction method and application thereof. The ELHK cell line is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on December 04, 2020, the address is the 5th floor of No.59 building, No.100, Middle Xianlie Road, Guangzhou City, Guangdong Province, the postal code is 510070, and the preservation number is GDMCC No: 61340. The Epinephelus lanceolatus head kidney cell line-ELHK cell line is obtained, the growth state is good, cell proliferation is stable, the cell morphology takes a fibroblast sample as the main morphology, continuous passage can be achieved (at present, the cells have already been transferred to more than 120 generations), ultralow-temperature cryopreservation and recovery can be achieved. The establishment of the cell line lays a foundation for related research of grouper germplasm resource preservation.

The invention belongs to the field of genetic engineering of aquatic animals, and relates to a CpG ODN sequence with immune enhancing activity on grass carp and application thereof. Disclosed is a CpG ODN sequence that has immune enhancing activity on grass carp, the sequence is the nucleotide sequence shown in SEQ ID NO: 1, it contains the oligodeoxynucleotide sequence of the CpG motif, and the sequence contains three 'AACGTT' repeats, and a 'TCGA' palindromic sequence. Through preliminary screening of grass carp head kidney mononuclear cells and grass carp kidney cell lines, the enhancement effect of CpG on the immune activity of grass carp was verified, and the CpG with the strongest immune enhancement activity on grass carp cells was screened out through its activation effect on genes related to signaling pathways ODN sequence. Through the challenge test, it was verified again that the CpG ODN sequence has a strong immune enhancing effect. The sequence can be prepared into a grass carp immune enhancer, an immune adjuvant or a vaccine, and can be popularized and applied in the prevention of grass carp diseases.

Provided is technology of external model construction of pseudosciaena crocea head kidney macrophage. The fish head kidney macrophage is horizontally and centrifugally separated by utilizing Percoll with 31% / 45% gradient and is cultured in an L15 compound culture medium under the condition that the temperature is 22 DEG C. Each liter of the culture medium contains 50ml fetal calf serum, 0.1 million UI penicillin / streptomycin, 0.05 million UI heparin, 10mmol 14-ethoxyl-piperazine ethanesulfonic acid (HEPES) and 0.5g glucose. The pH of the culture medium is 7.4. The method and the application have the advantages that (1) the proportion of the pseudosciaena crocea head kidney macrophage which is centrifugally separated by utilizing Percoll with 31% / 45% gradient in the separation cell is as high as 42%; (2) the purity of the adherence pseudosciaena crocea head kidney macrophage after liquid culture is as high as 90%; (3) the livability of the pseudosciaena crocea head kidney macrophage after one week culture is higher than 58%; and (4) a function checking experiment proves that LPS can remarkably affect the swallow function and breath eruption activity of the cultured pseudosciaena crocea head kidney macrophage.

The invention discloses a method for characterizing a microorganism phagocytosis function of macrophages of trachinotus ovatus. The trachinotus ovatus is dissected and separated, and the macrophages of the head kidney of the trachinotus ovatus are obtained and are extracted with a Percoll solution density gradient centrifugation method. The number of survived cells cultured in vitro is calculated with a trypan blue staining method, and the survival condition of the cells in one week is detected. The phagocytosis of the macrophages of the head kidney of the trachinotus ovatus on Nocardia seriolea, saccharomycetes and the like is determined through a phagocytosis experiment, so that the phagocytosis function of the macrophages is observed. An experimental result shows that the macrophages of the trachinotus ovatus can be extracted very well, and the survival condition and the phagocytosis condition of the macrophages of the trachinotus ovatus can be observed very well. A foundation is laid for further research of physiology, pathology and toxicology of the macrophages of the trachinotus ovatus.

The invention discloses a system for characterizing macrophage phagocytosis microorganisms of pompanos. The pompanos are dissected and separated through a surgical knife and tweezers to obtain heads and kidneys; the heads and the kidneys are abraded through a cell sieve to obtain cell suspension containing macrophages, and the macrophages of the heads and the kidneys are obtained through a Percoll solution density gradient centrifugal tube. The survival amount of cells cultured in vitro through a trypan blue dyeing manner is counted, and the survival condition in one week is detected. The system is used for carrying out a phagocytosis experiment, and phagocytosis conditions of nocardia, yeast and the like of the pompanos by the head and kidney macrophages of trachinotus ovatus are determined, and a phagocytosis function is observed. An experiment result of the system shows that the macrophages of the pompanos can be extracted very well, and the survival condition and the phagocytosis condition of the macrophages of the pompanos can be observed very well. The system disclosed by the invention lays a foundation for further researches on physiology, pathology and toxicology of the macrophages of the pompanos.

The invention aims to solve the problem that two IgM heavy chains of epinephelus coioides are difficult to recognize at the same time, and provides a preparation method of a monoclonal antibody capable of recognizing two IgM heavy chains of epinephelus coioides. A unique IgM prokaryotic expression vector is constructed; IgM recombinant protein is expressed and purified, the prepared monoclonal antibody can recognize 80kDa and 50kDa of two bands in epinephelus coioides serum at the same time, the monoclonal antibody can effectively mark B cells in epinephelus coioides head kidney slices, and afoundation is laid for further researching functions of two IgM heavy chains of epinephelus coioides.

The invention discloses a grass carp IFN-γ2 gene, which has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table. The invention also discloses a recombinant protein expressing the grass carp IFN-γ2 gene, which has the amino acid sequence shown in SEQ ID NO: 2 in the sequence table. The recombinant protein can promote the activation of grass carp head kidney macrophages and up-regulate the expression of immune-related genes in vitro. Injecting the recombinant protein into grass carp can significantly reduce the mortality rate of grass carp infected with Flavobacterium columnar, improve the immune protection rate of grass carp, regulate the immune index of grass carp, and enhance the ability of grass carp to resist bacterial infection.

The invention relates to an epinephelus coioides interferon IFNgamma2 gene and a protein encoded by the sameas well as application of the protein in preparing a novel immunopotentiator. According to the invention, an open reading frame of the IFNgamma2 gene is cloned from epinephelus coioides head-kidney total RNA by specific primers SEQ ID NO:4 and SEQ ID NO:5 by means of a method of a primer amplification gene segment and RACE overall length amplification. The nucleotide sequence is shown as SEQ ID NO:2. The amino acid sequence of encoded protein is shown as SEQ ID NO:1. An initial test shows that the epinephelus coioides interferon IFNgamma2 protein can induce relevant immunogene expression and can be developed to natural fish immunopotentiator or immunologic adjuvant.

The present invention provides a method for the isolation and primary culture of grass carp dendritic cells, comprising the following steps: Step 1, the separation of grass carp spleen and head kidney: the grass carp is anesthetized to death, the spleen and head kidney are taken out, and the attached connective tissue is removed and adipose tissue and soaked; step 2, preparation of grass carp spleen and head kidney single cell suspension: continuous sieve filtration to obtain cell suspension; step 3, separation of grass carp spleen and head kidney mononuclear cells: Ficoll separation, after centrifugation Absorb and wash the cells of the buffy coat; step 4, static culture: adjust the concentration of monocytes, and place them in an incubator for culture; step 5, enrichment and purification of grass carp dendritic cells: collect suspension cells after culturing for a period of time, and use Grass carp dendritic cells were purified by density gradient centrifugation. The method can effectively prepare a large amount of grass carp dendritic cells, and the purity of the obtained grass carp dendritic cells can reach more than 80 percent; the operation process is simple and clear, and the time consumption is short.

A method for preparing chromosomes of red-spotted grouper adult fish belongs to the field of cell biology, and it includes pre-treatment of materials, preparation of kidney cell suspension, hypotonic treatment, fixation, dripping and staining; the pre-treatment of materials is Using the phytohemagglutinin injection method in vivo, inject PHA at the base of the pectoral fin of the experimental fish at a dose of 15 μg / g fish body weight, and inject colchicine solution at a dose of 2 μg / g fish body weight after 24 hours, and after 2.5-3 hours , take out the head kidney. The present invention adopts the local method of one-time injection of phytohemagglutinin, increases the injection concentration at the same time, can obtain good effect after 24 hours, and saves time. In the present invention, due to the characteristics of tissue cells of the red-spotted grouper, it is difficult to obtain complete cells when the hypotonic treatment is performed according to the prior art. Therefore, the present invention creatively changes the prior art and obtains a clear chromosome picture.

The invention discloses a grass carp bacterial septicemia and red skin disease combined vaccine and a preparation method thereof, using β-propiolactone as an inactivator to develop safe and efficient grass carp Aeromonas hydrophila septicemia and Pseudomonas aeruginosa Bacterial erythroderma dual white oil inactivated vaccine can effectively control the occurrence of bacterial septicemia and erythroderma in grass carp. After immunizing grass carp, it can stimulate the expression of IgM, IL-1β, IFN, Mx, C3 and TLR3 immune-related genes in the spleen and head kidney tissue of the fish, and the vaccine can produce a relative immune protection rate of 80-90% for grass carp , the relative protection rate can still maintain 50% 1 year after immunization; the invention is easy to operate, safe and effective, and two diseases can be prevented by one immunization, which not only reduces the use of antibiotics and chemical drugs, but also reduces the impact on the breeding environment and ecological environment, etc. The pollution and destruction of the external environment ensure the food safety and water ecological safety of grass carp.

The invention discloses a method for improving the streptococcus iniae infection resisting capability of GIFT tilapia in high-density culture. The method comprises the steps of preparing of compound Chinese herbal medicine, adding, feeding and breeding management. Compared with the prior art, the method has the following advantages: (1) the immunological protection of the GIFT tilapia in high-density culture can be effectively improved, and the survival rate of GIFT tilapia infected with streptococcus iniae is improved; (2) the compound Chinese herbal medicine added in a feed belongs to environment-friendly feed additives, and has less negative effect on fish and water; (3) the blood and head-kidney immunological stress response can be effectively enhanced, and the liver stress injury is reduced; (4) the adopted technological steps of the method are simple, the Chinese herbal medicine is low, and easy to obtain and preserve, and can be well absorbed and utilized by a fish body, and excrement of the GIFT tilapia does not impact the water quality.

The invention discloses a system for characterizing macrophage phagocytosis microorganisms of pompanos. The pompanos are dissected and separated through a surgical knife and tweezers to obtain heads and kidneys; the heads and the kidneys are abraded through a cell sieve to obtain cell suspension containing macrophages, and the macrophages of the heads and the kidneys are obtained through a Percoll solution density gradient centrifugal tube. The survival amount of cells cultured in vitro through a trypan blue dyeing manner is counted, and the survival condition in one week is detected. The system is used for carrying out a phagocytosis experiment, and phagocytosis conditions of nocardia, yeast and the like of the pompanos by the head and kidney macrophages of trachinotus ovatus are determined, and a phagocytosis function is observed. An experiment result of the system shows that the macrophages of the pompanos can be extracted very well, and the survival condition and the phagocytosis condition of the macrophages of the pompanos can be observed very well. The system disclosed by the invention lays a foundation for further researches on physiology, pathology and toxicology of the macrophages of the pompanos.

The invention discloses a combined vaccine for bacterial septicemia and red skin disease of grass carps, and a preparation method of the combined vaccine. The safe and efficient two-tier white oil inactivated vaccine against aeromonas hydrophila septicaemia and pseudomonas aeruginosa red skin disease is developed by using beta-propiolactone as an inactivator, and can effectively control the occurrence of the bacterial septicemia and red skin disease of the grass carps. After the grass carps are immunized with the vaccine, the vaccine can stimulate the expression of IgM, IL-1beta, IFN, Mx, C3 and TLR3 immune-related genes in fish spleen and head kidney tissues. The vaccine can produce up to 80-90% relative immune protection rate for the grass carps; the relative protection rate can still bemaintained at 50% after immunization is performed for one year. The combined vaccine is simple to operate, is safe and effective, can prevent two diseases after being used for immunizing at a time, and not only reduces the use of antibiotics and chemical drugs, but also reduces the pollution and destruction of the external environments such as breeding environment and ecological environment, thusguaranteeing the food safety and water ecological security of the grass carps.

The invention discloses a pseudosciaena crocea head kidney cell line and a construction method thereof, relating to cell lines. The pseudosciaena crocea head kidney cell line is a pseudosciaena crocea head kidney cell line LYCK with the preservation number of CCTCC NO: C2013113. The construction method of the pseudosciaena crocea head kidney cell line comprises the steps of (1) preparing a cell culture medium; (2) constructing primary culture; (3) performing secondary culture. The constructed pseudosciaena crocea head kidney cell line is fibrous, can be subjected to continuous passage, and is sub-cultured for 70 generations; a large quantity of pseudosciaena crocea head kidney cell lines can be provided; the pseudosciaena crocea head kidney cell line can be directly applied to research of various pseudosciaena crocea immunity related functional genes. The pseudosciaena crocea head kidney cell line can be applied to important researches including cytobiology, molecular biology, virology, toxicology, oncology, genetics, immunology and the like.

The invention discloses the construction method and application of the grass carp matrix metalloproteinase inhibitory factor-2 gene, the encoded recombinant protein and its spatiotemporal expression map. The nucleotide sequence of the grass carp matrix metalloproteinase inhibitor-2 gene of the present invention is shown in SEQ ID NO:1. The amino acid sequence of the recombinant protein encoded by the nucleotide sequence is shown in SEQ ID NO:2. The matrix metalloproteinase inhibitor-2 gene of grass carp of the present invention is expressed in grass carp tissue and development process, and this gene is transcribed in a large amount in blood, mesonephros, head kidney, spleen, gill, intestine, heart, in brain, muscle, liver , There is a small amount of transcription in the skin, and the existence of a small amount of mRNA is detected in the fin rays. The gene was transcribed in a large amount from the caudal fin stage, and reached the maximum expression level on the 3rd day of fry. The invention also discloses the application of the grass carp matrix metalloproteinase inhibitor-2 gene and the coded recombinant protein in raising grass carp seedlings and as a feed additive for grass carp.

The invention discloses a method for characterizing a microorganism phagocytosis function of macrophages of trachinotus ovatus. The trachinotus ovatus is dissected and separated, and the macrophages of the head kidney of the trachinotus ovatus are obtained and are extracted with a Percoll solution density gradient centrifugation method. The number of survived cells cultured in vitro is calculated with a trypan blue staining method, and the survival condition of the cells in one week is detected. The phagocytosis of the macrophages of the head kidney of the trachinotus ovatus on Nocardia seriolea, saccharomycetes and the like is determined through a phagocytosis experiment, so that the phagocytosis function of the macrophages is observed. An experimental result shows that the macrophages of the trachinotus ovatus can be extracted very well, and the survival condition and the phagocytosis condition of the macrophages of the trachinotus ovatus can be observed very well. A foundation is laid for further research of physiology, pathology and toxicology of the macrophages of the trachinotus ovatus.