CN104450782A - Method of preparing silkworm model of hyperproteinemia - Google Patents
Method of preparing silkworm model of hyperproteinemia Download PDFInfo
- Publication number
- CN104450782A CN104450782A CN201410582543.9A CN201410582543A CN104450782A CN 104450782 A CN104450782 A CN 104450782A CN 201410582543 A CN201410582543 A CN 201410582543A CN 104450782 A CN104450782 A CN 104450782A
- Authority
- CN
- China
- Prior art keywords
- silkworm
- sequence
- hpl
- hyperproteinemia
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of medicines and discloses a method of preparing a silkworm model of hyperproteinemia and application of the method. The preparation method of the silkworm model of the hyperproteinemia comprises the following steps: recombining DNA molecules of a coded amino acid sequence HPL onto a silkworm chromosome for expressing and translating to obtain the silkworm model, wherein a silkworm Fib-H gene promoter, a silkworm Fib-H gene 5'-terminal signal peptide coding sequence, an Hpl sequence and a silkworm Fib-H gene 3'-terminal sequence are arranged from the 5' terminal to the 3' terminal of the coded amino acid sequence HPL, the Hpl sequence has the amino acid sequence as shown in SEQ ID NO:1. The method of preparing the silkworm model of hyperproteinemia is simple to operate, capable of quickly obtaining the silkworm model of hyperproteinemia, wherein the circulating blood total protein level with long time holding window of the stably inherited hyperproteinemia is higher than that of the normal control animals by over 50%.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of method preparing the animal model of hyperproteinemia.Especially a kind of method preparing the silkworm model of hyperproteinemia.
Background technology
Carbohydrate metabolism, lipid metabolism and protein metabolism are the animal basic nutrition metabolism comprising the mankind.Physiology and metabolic trouble research fully confirm that the glucose level in too high blood circulation or blood lipid level have detrimentally affect widely to the most animal bodies comprising the mankind, are serious metabolic troubles.Protein (amino acid) metabolic disturbance is also common metabolic trouble, comprise hyperproteinemia and hypoproteinemia, wherein hyperproteinemia refers to that clinical symptom occurs that protein in the recycle system (amino acid) horizontal abnormality raises, and hypoproteinemia refers to that clinical symptom occurs that protein in the recycle system (amino acid) horizontal abnormality reduces.Total serum protein and white protein increase, clinically be mainly seen in acute dehydration, hypoadrenocorticism, multiple myeloma, lymphoma, primary macroglobulinaemia, the autoimmune disorders such as systemic lupus erythematous, rheumatic fever, rheumatoid arthritis, and chronic inflammatory diseases and the infection such as tuberculosis, malaria, chronic schistosomiasis.Total serum protein and white protein reduce, and be common in major Liver disease, malnutrition, nephrotic syndrome, serious burn, the acute reason such as to lose blood greatly cause PD too much, and the chronic wasting disease such as severe tuberculosis, hyperthyroidism and malignant tumour.
The weight of metabolic disease clinical manifestation, the degree depending on metabolism disorder and the degree that organs in adult structure and fuction is destroyed.It should be noted that the metabolic disturbances such as sugar, fat and protein, usually influence each other and contact, causing vicious cycle sometimes.Various metabolic disease all can affect that whole body is respectively organized, organ, and many metabolic diseases can affect intelligence, grow and the mental status.And metabolic disease lacks radical cure method mostly at present.For this reason, protein (amino acid) metabolic trouble as sugar (carbohydrate) metabolism, fat (lipid) metabolic trouble, will be subject to biomedical great attention.
Protein metabolism take amino acid as core.First albumen in food all will be degraded to amino acid and could be utilized by body, and in body, albumen also first will be decomposed into amino acid and could continue oxygenolysis or conversion.Total free aminoacids can synthesize oneself protein, also can be converted into carbohydrate or lipid or synthesize other biological active substance, and also energy is released in oxidable decomposition.Total free aminoacids synthetic protein in human body is its main application, account for the proportion of utilization of 75%, and the energy that protein provides accounts for the 10-15% of needed by human body total energy.Amino acid also by nitrogenous compound important in special metabolism compound body, as neurotransmitter, purine, pyrimidine, phosphatide, porphyrin, coenzyme etc.The synthesis of phosphatide needs S-adenosylmethionine, and the amine that amino acid decarboxylase produces often has special role, if serotonin is neurotransmitter, lacks, depression, suicide easily occur; Histamine and anaphylaxis have close ties.Inside and outside cells in vivo, in liquid, all total free aminoacidss are called total free aminoacids storehouse, though 1% of the not enough total amino acid content of its content, can reflect the overview of body nitrogen metabolism.And protein in blood circulation and amino acid levels can reflect the complicated result of the entirety of body protein metabolism.
At present, heredopathia caused by disorder of amino acid catabolism has found more than 100 kinds, mainly comprises pku, cystinuria, imino-glycin uria disease, histidinemia, Hartnup disease, other hyperphenylalaninemias, hyper prolinemia, hyperlysinemia etc.Aminoacidopathy also claims aminoacidopathy (aminoacidopathy) or is called amino acid uria (aminoaciduria), the deficient that amino acid catabolic can be divided into block and Amino Acid Absorption movement system defect type.Aminoacidopathy is autosomal recessive inheritance and causes a disease, and wherein much has obvious nervous system abnormality, causes amino acid whose renal transport defect, or can cause Secondary cases nervous system damage.Hereditary tyrosinemia infant blood tyrosine content increases, and urine tyrosine also increases, and the amino acid such as blood methionine(Met) (methionine(Met)) also can increase.But due to serum protein concentrations too high and to cause or the relative disease that causes and harm are not also studied clear, therefore, inexpensive, stable, the strong animal model of repeatability is expected to for the research of hyperproteinemia brings hope.
Animal disease model has vital role in metabolic trouble pathogenesis and protective agents are developed and clinical treatment is studied.There are a large amount of hyperglycemia or hyperlipidemia animal model modeling method at present, various types of hyperglycemia or hyperlipidemia animal model can be provided for biological basic medical and clinical study, but also lack effective hyperproteinemia animal model, lack hyperproteinemia animal model modeling method.
The metabolic trouble animal model used at present is both at home and abroad not only the mammalss such as mouse in vertebrates, dog, pig and monkey, also comprises the invertebrates such as fruit bat, nematode.Because Mammals is as the disease model of internal metabolism, carry out extensive metabolism detection in the medicine primary dcreening operation stage and need at substantial financial resources, also can cause a series of ethics problem, and this has become a major reason of developed country's restriction drug development, also become the barrier hindering China to carry out external cooperation research and pharmaceutical prod trade.Therefore, use the invertebrates of low cost to substitute Mammals and carry out drug screening as animal model and evaluation has huge development prospect (Berger J:Preclinical testing on insects predicts human haematotoxicpotentials.Lab Anim 2009,43 (4): 328-332.).
Silkworm is the model invertebrate organism of distinct Chinese characteristics, is widely used in heredity and grows research.Silkworm, as the evaluating drug effect laboratory animal of the lethal research mode animal of metabolic trouble and drug candidate composition, has unique advantage compared to experiment in vitro and conventional fruit bat and Mammals model animals.Silkworm is current genetic development and pathological research one of insect the most clearly, and its nearly thousand genetic backgrounds of growing mutantion line and disease model are clear.Silkworm size to fit, it is convenient to dissect, and pathology sample is directly perceived; Safely, reliably can carry out hemolymph injection and oral cavity injection, mammiferous intravenous injection and oral administration treatment effect can be reached; Compare fruit bat, silkworm activity torpescence, there is operability more better than drosophila embryos, also there is the heredity more complicated than nematode and developmental regulation mechanism, be well suited for carrying out toxicity, heredity and growing research; As the insect of unique domestication, silkworm leaves the mankind and cannot survive separately and produce offspring, and can not escape in experiment; Silkworm is the aseptic raising of complete available artificial diet in laboratory, and utensil, envrionment conditions are simply controlled, and biological safety is good.Adopt silkworm etc. to replace high laboratory animal without vertebra model animals laboratory facilities, alleviate or reduce the pain and uneasiness that cause to higher animal, decreasing animal welfare dispute, is also the core place of international animal ethics welfare 3R principle.And the pharmacological experiment research on organ level confirms, middle intestines, the fatty body of silkworm are equivalent to mammiferous enteron aisle and liver respectively; The absorbing state of the exogenous materials such as microbiotic in enteron aisle is also closely similar in silkworm and Mammals; The drug metabolism mechanism identical with Mammals is there is in silkworm.As can be seen here, use silkworm to build disease animal model and there is huge development potentiality.And also there is no the silkworm model of hyperproteinemia at present.
Summary of the invention
An object of the present invention is to provide a kind of method preparing the silkworm model of the hyperproteinemia of genetic stability, the DNA molecular of encoding amino acid sequence HPL is recombinated on chromosome of mulberry silkworm, express and translate and get final product; Wherein said encoding amino acid sequence HPL 5' holds the nucleotide sequence to 3' end to be followed successively by silkworm Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide-coding sequence, Hpl sequence and silkworm Fib-H gene 3' terminal sequence, and described Hpl sequence has aminoacid sequence as shown in SEQ ID NO:1.
In some embodiments, described aminoacid sequence HPL has aminoacid sequence as shown in SEQ ID NO:2.
In some embodiments, shown in described coding SEQ ID NO:2, the DNA molecular of aminoacid sequence has the nucleotide sequence as shown in SEQ ID NO:3.
In some embodiments, the described DNA molecular by encoding amino acid sequence HPL is incorporated into transposon vector chromosome of mulberry silkworm being specially and building and comprise the DNA molecular of encoding amino acid sequence HPL, is imported in Eggs of Silkworm by transposon vector.
Second object of the present invention is to provide a kind of transposon vector with the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:2.
In some embodiments, described transposon vector is PB-Hpl.
In some preferred embodiments, described transposon vector has nucleotide sequence as shown in SEQ ID NO:4.
3rd object of the present invention is to provide a kind of silkworm model of hyperproteinemia, and the silkworm model of described hyperproteinemia is prepared by above-mentioned preparation method.
Compared with prior art, the method of the silkworm model of preparation hyperproteinemia of the present invention is simple to operate, and the hyperproteinemia that can obtain genetic stability is rapidly held time the silkworm model of the long circulating total protein levels of window higher than the hyperproteinemia of Normal control animals more than 50%.The silkworm animal pattern tissue deterioration of the hyperproteinemia that preparation method of the present invention prepares and tissue remodeling slack-off and abnormal, heteroplasia rate and mortality ratio significantly raise.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows and turns Hpl sequence carrier structure schematic diagram, wherein the transposon vector of A to be recombination Hpl, B be recombination Hpl;
Fig. 2 shows the difference figure of Hpl silkworm and positive control silkworm; Wherein scheme the fluorogram that A is silkworm chrysalis head, figure B is condition diagram of cocooing; In each figure, CK0 is the negative control of normal silkworm, and CK turns not contain Hpl sequence but the empty carrier positive control silkworm having ERFP, and Hpl is the silkworm turning recombination Hpl.
Embodiment
Below in conjunction with the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The sericterium of silkworm larva is the face tissue of specialization, single-minded synthesis silk protein, it is the most powerful animal tissues of known protein synthesis capacity, also be the vigorous tissue of a kind of quick growth similar to tumour cell, metabolism, it is weight rapid growth nearly 10,000 times within 3.5 weeks larval stages, and synthesis exceedes the silk protein of organism own wt 30%.Silkworm larva can spue after maturation the overwhelming majority synthesize and be stored in sericterium protein formation silk cocoon cocoon layer (cocoon shell), and by autophagocytosis, sericterium tissue is dissociated in metamorphosis process subsequently, the protein of the residue silk protein in sericterium chamber and sericterium tissue is together discharged into circulating lymph.Further, silkworm is by a kind of metabolism of metabolizing tissue's fatty body of similar human liver, and the free protein in circulating lymph is progressively decomposed into amino acid by collaborative hemolymph, then carries out oxygenolysis or trans-utilization further.
Current silkworm biological study clearly, domestic natural silk gland by anterior division of silkgland, middle division of silkgland and posterior division of silkgland three part form.Anterior division of silkgland is the secreted in vitro passage of silk protein, does not participate in the synthesis of silk protein.The outer sericine of middle division of silkgland synthesis silk.Core-the fibroin protein of posterior division of silkgland synthesis silk protein fiber.Wherein, the silk fibroin of posterior division of silkgland synthesis contains heavy chain (Fib-H), light chain (Fib-L) and P25 tri-kinds of protein component (Sprague.1975; Couble et al.1983; Yamaguchi etal.1989), three forms complex body, as the fundamental structural unit (Inoue et al., 2000) of fibroin protein with the mol ratio of 6:6:1.Wherein Fib-H and Fib-L is interconnected with disulfide linkage by Cys residue, and P25 is then combined with Fib-H/Fib-L complex body with non covalent bond-hydrophobic interaction.Wherein disulfide linkage connects Fib-H/Fib-L complex body has very important effect (Takei et al, 1987) for fibroin protein at silk gland cell transmitter loss with to lumen of gland secretion.
The invention provides a kind of method preparing the silkworm model of hyperproteinemia, the DNA molecular of encoding amino acid sequence HPL is recombinated on chromosome of mulberry silkworm, express and translate and get final product; Wherein said encoding amino acid sequence HPL 5' holds the nucleotide sequence to 3' end to be followed successively by silkworm Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide-coding sequence, Hpl sequence and silkworm Fib-H gene 3' terminal sequence, and described Hpl sequence has aminoacid sequence as shown in SEQ ID NO:1.
Encoding amino acid sequence HPL described in preparation method of the present invention is highly similar to bombyx mori silk fibroin heavy chain Fib-H, the DNA molecular of encoding amino acid sequence HPL is recombinated on chromosome of mulberry silkworm, described encoding amino acid sequence HPL is after silkworm expression in vivo, destroy the normal formation of Fib-H/Fib-L complex body, in control sericterium histocyte, fibroin protein is to lumen of gland and secreted in vitro, induction silk fibroin is trapped in silk gland cell or lumen of gland, silkworm is utilized to pupate decomposing from row degradation of sericterium tissue in metamorphosis process, the a large amount of protein of quick release is to circulating lymph, protein in hemolymph concentration is caused to increase severely, protein content in rapid rising hemolymph, final formation hyperproteinemia environment, and maintain almost whole pupa development period.
Wherein, described Fib-H gene promoter (Fib-H promotor) is for starting the expression of Hpl nucleotide sequence, the 5' end signal peptide sequence of described silkworm Fib-H gene is for ensureing that the HPL amino acid residue sequence translated has by Bombyx mori posterior silkgland emiocytosis function, the 3' terminal sequence of described Fib-H gene, with the Fib-H gene signal peptide sequence of upstream together with promoter sequence, Hpl sequence alterations is become the restructuring Hpl gene can expressed at silkworm posterior silkgland cells, and can secrete silk gland cell.Have 11 Cys residues in described Hpl sequence, also comprise a large amount of iterons, and in described Hpl sequence, Ala, Ser and Gly amino acid residue content is high, Ala, Ser and Gly ratio is up to 87.53%.
In some embodiments, described aminoacid sequence HPL has aminoacid sequence as shown in SEQ ID NO:2.
Due to the degeneracy of codon, the DNA molecular of a variety of aminoacid sequence HPL of the present invention that can encode can be there is.
In some embodiments, shown in described coding SEQ ID NO:2, the DNA molecular of aminoacid sequence has the nucleotide sequence as shown in SEQ ID NO:3.
Those skilled in the art can obtain step according to general silkworm transgeneic procedure and transgenic silkworm, recombinate on chromosome of mulberry silkworm, prepare the silkworm model of the hyperproteinemia of genetic stability by the DNA molecular of encoding amino acid sequence HPL.
In some embodiments, the described DNA molecular by encoding amino acid sequence HPL is incorporated into transposon vector chromosome of mulberry silkworm being specially and building and comprise the DNA molecular of encoding amino acid sequence HPL, is imported in Eggs of Silkworm by transposon vector.
Wherein, described method transposon vector being imported Eggs of Silkworm is preferably microinjection.
Further, in some specific embodiments, following step is taked to be incorporated on chromosome of mulberry silkworm by the DNA molecular of encoding amino acid sequence HPL:
1, transposon vector is built
Hold at the upstream 5' of artificial DNA sequence Hpl and connect silkworm Fib-H gene promoter and silkworm Fib-H gene 5' end signal peptide-coding sequence, hold the 3' terminal sequence connecting silkworm Fib-H gene to obtain recombination Hpl at artificial DNA sequence downstream 3'.Piggybac-3 × P3DsRedaf plasmid (sequence is as shown in SEQ ID NO:5) of Artificial promoters 3 × P3 of the family's the inactive state of the silkworm before it sheds its skin containing 3 series connection and nervous system specific transcription factor PAX-6 binding sequence composition is carried out AscI and FseI double digestion respectively with the Hpl sequence of synthetic.Piggybac-3 × P3DsRedaf plasmid after being cut by enzyme and Hpl sequence, with being connected, form transposon vector PB-Hpl.Described recombination Hpl and transposon vector PB-Hpl structural representation are shown in Fig. 1.
2, the silkworm of hyperproteinemia is obtained
(1), in the environment that aseptic and free nucleic acid pollutes, micro-injection method injects the transposon vector plasmid of 4000ng-5000ng to every silkworm seed;
(2) after injection, silkworm seed non-toxic material envelope is coated with injection orifice, then in gnotobasis, with 26 DEG C-27 DEG C, 75%-80% relative humidity, LD 12h:12h light system protection silkworm seed to hatching, after hatching larva mulberry leaf or artificial diet 25 DEG C-29 DEG C, 75%-80% relative humidity, natural light condition raise, continue humiture and photoenvironment between larval stage after pupating and protect to adult eclosion;
(3) silkworm moth selfing or after backcrossing after sprouting wings, after institute's spawning and hatching, larva mulberry leaf or artificial diet are raised, and the larva 3-5 length of time or pupa time are investigated under fluorescent microscope, and eye presents the individuality of red fluorescence, individual as transgenosis G1 generation;
(4) transgenosis G1 is selected to present the individual subculture of red fluorescence, to G6 for the rear silkworm namely obtaining the hyperproteinemia of genetic stability for the eye of individual larva or pupa.
Present invention also offers a kind of transposon vector with the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:2.
In some embodiments, transposon vector of the present invention comprises the Artificial promoters 3 × P3 with family's the inactive state of the silkworm before it sheds its skin of 3 series connection and nervous system specific transcription factor PAX-6 binding sequence composition, for control ERFP reporter gene.
In some embodiments, described transposon vector be PB-Hpl comprise PB transposase recognition sequence piggyBac3'LTR and piggyBac5'LTR, Artificial promoters 3 × P3, ERFP reporter gene, SV40 polyadenylic acid add tailer sequence, recombination Hpl; Wherein, described PB transposase recognition sequence lays respectively at the TTAA site on the left end of the piggyBac3'LTR on transposon vector and the right-hand member of piggyBac5'LTR; Be followed successively by between PB transposase recognition sequence piggyBac3'LTR and piggyBac5'LTR Artificial promoters 3 × P3, ERFP reporter gene, SV40 polyadenylic acid add tailer sequence and recombination Hpl.
In some preferred embodiments, described transposon vector has nucleotide sequence as shown in SEQ ID NO:4.
Present invention also offers the silkworm model of the hyperproteinemia of the genetic stability that preparation method of the present invention prepares.
With the silkworm that normally weaves silk for contrast, the silkworm model hyperproteinemia of the hyperproteinemia that the present invention prepares window of holding time is long, accounts for about 15% of life cycle.And the silkworm animal tissues of hyperproteinemia that the present invention prepares degenerates and tissue remodeling is slack-off and abnormal, heteroplasia rate and mortality ratio significantly raise.
Wherein, hyperproteinemia level of the present invention refers to circulating total protein levels during pupa time 4d to 11d higher than Normal control animals more than 50%; Circulating Determination of Free Amino Acids during pupa time 4d to 9d higher than Normal control animals more than 50%.
Heteroplasia rate of the present invention refers to that development by metamorphosis is abnormal, particularly pupa to adult abnormal and interior tissue sexual gland, fatty body morphological development slack-off with exception, the tissue development impact of hyperproteinemia and lethal model Effect study can be applicable to.
The dead individuals that high mortality of the present invention occurs during being the pupa development of hyperproteinemia initiation significantly raises, and the pupa development phase, general mortality rate was more than 70%.
The silkworm animal pattern tissue deterioration of the hyperproteinemia that preparation method of the present invention prepares, tissue remodeling is slack-off and abnormal, heteroplasia rate and mortality ratio significantly raise, the different steps of growing, overcomes by the drug screening giving different pharmaceutical or give different concns the medicine that the heteroplasia in silkworm pupa stage and the medicine of lethality and screening improve circulating Proteometabolism activity.Therefore the silkworm model that present invention also offers the hyperproteinemia that preparation method of the present invention prepares overcomes the heteroplasia in silkworm pupa stage and the medicine of lethality and screening in screening and improves application in the medicine of circulating Proteometabolism activity.
The silkworm model of the hyperproteinemia that preparation method of the present invention prepares pupa time circulating total protein levels significantly raise, cause metabolizing tissue's fat body cells impaired, performance fatty body rebuilding course is significantly postponed with pupa development abnormal, and mortality ratio significantly increases.The different steps of growing, make the reparation of fatty body function by the drug screening giving different pharmaceutical or give different concns, rebuilding course recovers normal medicine.Therefore the silkworm model that present invention also offers the hyperproteinemia that preparation method of the present invention prepares is screening the application improved in the medicine of fatty body reconstruction function.
In order to understand the present invention further, below in conjunction with embodiment, method provided by the invention is described in detail.
The preparation of the silkworm model of embodiment 1, hyperproteinemia
Connect promotor and the 5' end signal peptide-coding sequence of silkworm Fib-H gene in the upstream of artificial DNA sequence Hpl, the 3' terminal sequence that downstream connects silkworm Fib-H gene obtains recombination Hpl, and its nucleotide sequence is as shown in SEQ ID NO:3.
Piggybac-3 × P3DsRedaf the plasmid (sequence is as shown in SEQ ID NO:5) of Artificial promoters 3 × P3 family's the inactive state of the silkworm before it sheds its skin containing 3 series connection and nervous system specific transcription factor PAX-6 binding sequence formed and the Hpl sequence of synthetic carry out AscI and FseI double digestion (Hpl introduces AscI and FseI restriction enzyme site at two ends when synthetic) respectively.Piggybac-3 × P3DsRedaf plasmid after enzyme is cut and HP1 sequence T4 ligase enzyme 16 DEG C of reaction overnight.Then product is carried out electrophoresis detection, and the Hpl band cut by enzyme carries out purifying recovery, and deliver to company's order-checking.What qualification sequence was correct is transposon vector PB-Hpl, and its nucleotide sequence is as shown in SEQ ID NO:4.
Concrete schematic diagram as shown in Figure 1.The transposon vector of A to be recombination Hpl, B be recombination Hpl in figure.
Obtain step according to general silkworm transgeneic procedure and transgenic silkworm, recombination Hpl sequence is incorporated on silkworm No. 7 karyomit(e)s, obtains Hpl silkworm.To turn not containing Hpl sequence but to have the empty carrier silkworm of ERFP for (positive) contrast, carry out Fluirescence observation to the silkworm chrysalis head of the Hpl silkworm obtained, observe the situation of cocooing, result as shown in Figure 2 simultaneously.In figure, A is the fluorescence picture of silkworm chrysalis head, and B is the situation of cocooing.In each figure, CK0 is the negative control of normal silkworm, and CK turns not contain Hpl sequence but the empty carrier positive control silkworm having ERFP, and Hpl is the silkworm turning recombination Hpl.
Ruddiness fluorescence is all presented by the eye of the embryo of the visible Hpl silkworm of Fig. 2 result and positive control silkworm, larva, pupa and adult, but Hpl silkworm normally can not weave silk and cocoon, occur exarate pupa or knot thin shelled cocoon, and positive control silkworm equally with the normal silkworm of negative control can weave silk and bears normal cocoon.
The preparation of the silkworm model of embodiment 2, hyperproteinemia
Connect promotor and the 5' end signal peptide-coding sequence of silkworm Fib-H gene in the upstream of artificial DNA sequence Hpl, the 3' terminal sequence that downstream connects silkworm Fib-H gene obtains recombination Hpl, and its nucleotide sequence is as shown in SEQ ID NO:3.
Piggybac-3 × P3DsRedaf the plasmid (sequence is as shown in SEQ ID NO:5) of Artificial promoters 3 × P3 family's the inactive state of the silkworm before it sheds its skin containing 3 series connection and nervous system specific transcription factor PAX-6 binding sequence formed and the Hpl sequence of synthetic carry out AscI and FseI double digestion (Hpl introduces AscI and FseI restriction enzyme site at two ends when synthetic) respectively.Piggybac-3 × P3DsRedaf plasmid after enzyme is cut and Hpl sequence T4 ligase enzyme 16 DEG C of reaction overnight.Then product is carried out electrophoresis detection, and the Hpl band cut by enzyme carries out purifying recovery, and deliver to company's order-checking.What qualification sequence was correct is transposon vector PB-Hpl, and its nucleotide sequence is as shown in SEQ ID NO:4.
Concrete schematic diagram as shown in Figure 1.The transposon vector of A to be recombination Hpl, B be recombination Hpl in figure.
Obtain step according to general silkworm transgeneic procedure and transgenic silkworm, recombination Hpl sequence is incorporated on silkworm No. 16 karyomit(e)s, obtains Hpl silkworm.To turn not containing Hpl sequence but to have the empty carrier silkworm of ERFP for (positive) contrast, carry out Fluirescence observation to the silkworm chrysalis head of the Hpl silkworm obtained, observe the situation of cocooing, result is with embodiment 1 simultaneously.
The preparation of the silkworm model of embodiment 3, hyperproteinemia
Connect promotor and the 5' end signal peptide-coding sequence of silkworm Fib-H gene in the upstream of artificial DNA sequence Hpl, the 3' terminal sequence that downstream connects silkworm Fib-H gene obtains recombination Hpl, and its nucleotide sequence is as shown in SEQ ID NO:3.
Piggybac-3 × P3DsRedaf the plasmid (sequence is as shown in SEQ ID NO:5) of Artificial promoters 3 × P3 family's the inactive state of the silkworm before it sheds its skin containing 3 series connection and nervous system specific transcription factor PAX-6 binding sequence formed and the Hpl sequence of synthetic carry out AscI and FseI double digestion (Hpl introduces AscI and FseI restriction enzyme site at two ends when synthetic) respectively.Piggybac-3 × P3DsRedaf plasmid after enzyme is cut and HP1 sequence T4 ligase enzyme 16 DEG C of reaction overnight.Then product is carried out electrophoresis detection, and the Hpl band cut by enzyme carries out purifying recovery, and deliver to company's order-checking.What qualification sequence was correct is transposon vector PB-Hpl, and its nucleotide sequence is as shown in SEQ ID NO:4.
Concrete schematic diagram as shown in Figure 1.The transposon vector of A to be recombination Hpl, B be recombination Hpl in figure.
Obtain step according to general silkworm transgeneic procedure and transgenic silkworm, recombination Hpl sequence is incorporated on silkworm No. 28 karyomit(e)s, obtains Hpl silkworm.To turn not containing Hpl sequence but to have the empty carrier silkworm of ERFP for (positive) contrast, carry out Fluirescence observation to the silkworm chrysalis head of the Hpl silkworm obtained, observe the situation of cocooing, result is with embodiment 1 simultaneously.
Claims (8)
1. prepare a method for the silkworm model of hyperproteinemia, the DNA molecular of encoding amino acid sequence HPL is recombinated on chromosome of mulberry silkworm, express and translate and get final product; Wherein said encoding amino acid sequence HPL5' holds the nucleotide sequence to 3' end to be followed successively by silkworm Fib-H gene promoter, silkworm Fib-H gene 5' end signal peptide-coding sequence, Hpl sequence and silkworm Fib-H gene 3' terminal sequence, and described Hpl sequence has aminoacid sequence as shown in SEQ ID NO:1.
2. method according to claim 1, described aminoacid sequence HPL has aminoacid sequence as shown in SEQ ID NO:2.
3. method according to claim 1, shown in described coding SEQ ID NO:2, the DNA molecular of aminoacid sequence has the nucleotide sequence as shown in SEQ ID NO:3.
4. method according to claim 1, the described DNA molecular by encoding amino acid sequence HPL is incorporated on chromosome of mulberry silkworm, is specially the transposon vector building and comprise the DNA molecular of encoding amino acid sequence HPL, is imported in Eggs of Silkworm by transposon vector.
5. one kind has the transposon vector of the DNA molecular of aminoacid sequence shown in coding SEQ ID NO:2.
6. carrier according to claim 5, it is characterized in that, described transposon vector is PB-Hpl.
7. carrier according to claim 5 or 6, is characterized in that, described transposon vector has nucleotide sequence as shown in SEQID NO:4.
8. the silkworm model of the hyperproteinemia that the method described in claim 1-4 any one prepares.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410582543.9A CN104450782B (en) | 2015-01-07 | 2015-01-07 | Method of preparing silkworm model of hyperproteinemia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410582543.9A CN104450782B (en) | 2015-01-07 | 2015-01-07 | Method of preparing silkworm model of hyperproteinemia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450782A true CN104450782A (en) | 2015-03-25 |
| CN104450782B CN104450782B (en) | 2017-01-25 |
Family
ID=52897531
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410582543.9A Expired - Fee Related CN104450782B (en) | 2015-01-07 | 2015-01-07 | Method of preparing silkworm model of hyperproteinemia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450782B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011172A (en) * | 2016-05-24 | 2016-10-12 | 苏州大学 | Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland |
| CN111793643A (en) * | 2020-07-17 | 2020-10-20 | 重庆西蚕生物技术研究院有限公司 | Silkworm fibroin heavy chain expression system for expressing target protein distributed in silk fibroin and sericin, preparation method and application |
| CN116548388A (en) * | 2023-06-29 | 2023-08-08 | 细胞生态海河实验室 | Preparation method of transgenic zebra fish model for marking hematopoietic stem/progenitor cell cycle |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1912116A (en) * | 2006-07-14 | 2007-02-14 | 西南大学 | Method for mass expressing external protein using domestic silk core protein heavy chain promoter |
| CN104212833A (en) * | 2013-05-30 | 2014-12-17 | 江苏大学 | Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo |
-
2015
- 2015-01-07 CN CN201410582543.9A patent/CN104450782B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1912116A (en) * | 2006-07-14 | 2007-02-14 | 西南大学 | Method for mass expressing external protein using domestic silk core protein heavy chain promoter |
| CN104212833A (en) * | 2013-05-30 | 2014-12-17 | 江苏大学 | Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011172A (en) * | 2016-05-24 | 2016-10-12 | 苏州大学 | Preparation method of bombyx mori capable of synthesizing and secreting hydrophilic sericin on posterior division of silkgland |
| CN111793643A (en) * | 2020-07-17 | 2020-10-20 | 重庆西蚕生物技术研究院有限公司 | Silkworm fibroin heavy chain expression system for expressing target protein distributed in silk fibroin and sericin, preparation method and application |
| CN116548388A (en) * | 2023-06-29 | 2023-08-08 | 细胞生态海河实验室 | Preparation method of transgenic zebra fish model for marking hematopoietic stem/progenitor cell cycle |
| CN116548388B (en) * | 2023-06-29 | 2023-10-10 | 细胞生态海河实验室 | Preparation method of transgenic zebra fish model for marking hematopoietic stem/progenitor cell cycle |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450782B (en) | 2017-01-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jozefiak et al. | Insect proteins as a potential source of antimicrobial peptides in livestock production. A review | |
| Wharton | A functional biology of nematodes | |
| Hall et al. | Stages of embryonic development in the Atlantic cod Gadus morhua | |
| Hamblin et al. | Non‐mammalian hosts and photobiomodulation: do all life‐forms respond to light? | |
| Chen et al. | Combined analysis of silk synthesis and hemolymph amino acid metabolism reveal key roles for glycine in increasing silkworm silk yields | |
| Hensley et al. | A convenient dry feed for raising zebrafish larvae | |
| CN104450782B (en) | Method of preparing silkworm model of hyperproteinemia | |
| CN103911383B (en) | Be suitable for transformation human acid fibroblast growth factor gene that domestic natural silk gland expresses and expression system and application | |
| CN105409846A (en) | Culture method for goldfish | |
| Anadón | Functional histology: the tissues of common coleoid cephalopods | |
| Ma et al. | Jaw malformation of hatchery reared golden pompano T rachinotus ovatus (L innaeus 1758) larvae | |
| Chen et al. | Induced hyperproteinemia and its effects on the remodeling of fat bodies in silkworm, Bombyx mori | |
| Chen et al. | Upper thermal tolerance of wild‐type, domesticated and growth hormone‐transgenic coho salmon Oncorhynchus kisutch | |
| CN108642059A (en) | Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application | |
| Kozol et al. | Novel husbandry practices result in rapid rates of growth and sexual maturation without impacting adult behavior in the blind Mexican cavefish | |
| CN105861515A (en) | Reconstructed human serum albumin gene suitable for cultivated silk gland expression and expression system and application thereof | |
| Mukai et al. | Development of sensory organs and changes of behavior in larvae of the sutchi catfish, Pangasianodon hypophthalmus | |
| Masuda | The critical role of docosahexaenoic acid in marine and terrestrial ecosystems: from bacteria to human behavior | |
| CN103766255A (en) | Method for breeding finless eels | |
| CN109528742A (en) | Pharmaceutical applications of the echinacoside as treatment hypoxic ischemic encephalopathy of newborn | |
| Dreon et al. | Biochemical composition, tissue origin and functional properties of egg perivitellins from Pomacea canaliculata | |
| KR101568467B1 (en) | Feed compositon for culturing fry of eel | |
| CN103920144B (en) | The new opplication of the deoxyribonuclease I of recombined human | |
| Duarte et al. | Ontogeny and embryonic description of Betta splendens, Perciformes (Regan, 1910) | |
| CN104542507B (en) | Preparation method and application of silkworm model of hyperproteinemia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170125 Termination date: 20200107 |