CN104479016A - Method for preparing antibodies through DNA tattooing immunization - Google Patents
Method for preparing antibodies through DNA tattooing immunization Download PDFInfo
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- CN104479016A CN104479016A CN201410857000.3A CN201410857000A CN104479016A CN 104479016 A CN104479016 A CN 104479016A CN 201410857000 A CN201410857000 A CN 201410857000A CN 104479016 A CN104479016 A CN 104479016A
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Abstract
The invention discloses a method for preparing antibodies through DNA tattooing immunization. The method comprises the steps of establishing antigen protein expression vectors, wherein cDNA fragments of target genes are extracted from host cell genomes in a reverse transcription PCR amplification mode and then cloned into the expression vectors; preparing a large amount of purified plasmid DNA through escherichia coli; carrying out plasmid DNA immunization, wherein the plasmid DNA is injected into the animal cuticle in a tattooing mode for expression and immunization; separating and purifying the antibodies, wherein hybridomas are screened, monoclonal antibodies are separated, and serums are purified and collected to extract and purify polyclonal antibodies. A traditional method adopting peptide chain and protein immunization is replaced with the method adopting a DNA plasmid immune animal, so that the defects that in a traditional antibody production method, antibody titer is low, specificity is poor and natural epitopes can not be recognized are overcome.
Description
Technical field
The present invention relates to antibody production techniques field, be specifically related to a kind of DNA of utilization and tatoo the method for passing immunization.
Background technology
Antibody refers to that the immunity system of body is under antigenic stimulation, that the plasmocyte be divided into by bone-marrow-derived lymphocyte or memory cell prolifera produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding.Mainly be distributed in serum, be also distributed in tissue juice and exocrine secretion.1964, the World Health Organization holds ad hoc meeting, to there is antibody activity and the sphaeroprotein relevant to antibody is referred to as immunoglobulin (Ig) (Ig), as myeloma protein, the abnormal immunoglobulin existed in the patients serum such as macroglobulinemia, cryoglobulinemia and the naturally occurring immunoglobulin (Ig) subunit of normal people etc.
In conventional antibodies production method, immune animal mainly uses two para-immunity original matter, and one is for the peptide molecule designed by antibody target spot; Equations of The Second Kind is the recombinant protein intestinal bacteria or eukaryotic expression.And some insoluble problems of consequent antibody long-term existence.There is immunogenicity difference in such as polypeptide immune, and itself lacks the shortcoming of spatial conformation, and the antibody thus produced often avidity is low, can not be combined with native protein.And Successful vaccine acceptance is low, be generally less than 50%.And the recombinant protein of expressing in bacterium does not have intrinsic the folding of eukaryotic cell albumen, conformation and modification, the antibody thus produced is often also undesirable.Eukaryotic expression system can produce the antigen protein with native conformation, but because expression amount is low and cost of manufacture is high, is also unsuitable for a large amount of use.And also can change its proterties and conformation in antigen protein and immunological adjuvant mixing process, reduce the immunogenicity of antigen, cause the titre of antibody and specificity not high.
Summary of the invention
In order to overcome the deficiencies in the prior art, a kind of DNA of utilization is the object of the present invention is to provide to tatoo the method for passing immunization, the method of DNA plasmid immunizations animal is adopted to replace traditional peptide chain and Western Immuno, solve the antibody titers existed in conventional antibodies production method low, poor specificity and the defect of native antigenic epitopes can not be identified.
For solving the problem, the technical solution adopted in the present invention is as follows:
Utilize DNA to tatoo the method for passing immunization, the method comprises
1) step of antigen protein expression vector is built: the mode that the cDNA segment of goal gene is increased by reverse transcription PCR recalled from host cell gene group, be then cloned in expression vector;
2) plasmid DNA of purifying is prepared: utilize intestinal bacteria to prepare the plasmid DNA of purifying in a large number;
3) Plasmid DNA immunization: the mode of plasmid DNA being tatooed is injected in animal cuticle carries out expressing and immunity;
4) abstraction and purification antibody: adopt hybridoma screening monoclonal antibody separation and purification or collect serum and carry out polyclonal antibody extraction purification.
Particularly, step 1) middle employing pcDNA3.1 eukaryotic expression vector, goal gene is inserted between the NheI/EcorI double enzyme site on expression vector; The carrier built correctly is inserted in the protein expression reading frame on carrier with the exactness and sequence of guaranteeing goal gene sequence through sequence verification.
Particularly, step 2) be specially: the carrier built is transferred in DH5 α e. coli host cell and increases; Increased rear Isolation and purification plasmid.
Particularly, the present invention adopt Qiagen without intracellular toxin plasmid extraction kit Isolation and purification plasmid.
Particularly, described step 3) comprise the following steps:
S1: select animal back leg side to be position of tatooing, animal is anaesthetized, enters after narcosis until animal, electric shaver-for women removes the hair of need tatoo position and periphery, and cleans out;
S2: DNA plasmid good for purifying is applied in 1cm
2the position of the above-mentioned rejecting hair in scope, tatoos to it with syringe needle, complete after tatooing and coat pain relieving creme with clean cotton swab at skin surface of tatooing, and every two weeks repeats to tatoo immunity once;
S3: get blood between duration of immunity and be ELISA and Western blot and detect;
S4: until ELISA titre reach more than 1:8000 and be greater than negative control 3 standard deviations and Westernblot detects and sees correct positive band time carry out the fusion of splenocyte or the collection of serum and purifying; Once reinforced immunological is carried out at the DNA plasmid of fusion or purified blood serum first 3 days muscle hemostasis purifying.
Particularly, above-mentioned steps 5) described in animal be selected from mouse or New Zealand white rabbit.
Particularly, step 4) in from serum the method for Isolation and purification antibody be in salting-out process, column chromatography purification, affinitive layer purification or ion exchange chromatography one or more combine.
Compared to existing technology, beneficial effect of the present invention is:
1., in method of the present invention, DNA plasmid enters after in epithelial cell, cell protein machine synthetic antigen protein, and the albumen of synthesis does not exist the molecular folding in prokaryotic expression, the problem such as conformation and glycosylation modified defect; Antigen protein can be long-time, expresses constantly in epithelial cell;
2. in method of the present invention, common apparatus of tatooing is adopted the DNA plasmid of some amount to be entered the intracutaneous of animal by secondary puncture injection up to ten thousand, and the process of the antigen presenting cell adopting the antigen that produces of mode of repeatedly immunity abundant in skin histology and transmission, activate the function of T th cell and then activating B cell secretory antibody, thus produce avidity and the good high quality antibody of specificity tool;
3. stimulate the antibody produced can identify the albumen of native conformation by method immunogen of the present invention, thus in reagent for clinical diagnosis and antibody drug research and development, there is important using value;
4. utilize the plasmid immunizations animal of encode antigenic proteins gene in method of the present invention, antigen protein can be long-time, and high level is expressed in epithelial cell, and Successful vaccine acceptance is high, can reach more than 80%;
5. in method of the present invention, immunizing antigen is plasmid-encoded natural protein, therefore the antibody produced can identify albumen natural epitopes, therefore there is very high using value in clinical detection and scientific research etc., immunohistochemical methods (IHC) can be applied in, Immunofluorescence test (ICC), flow cytometer (FACS), in the multinomial detections such as enzyme linked immunological (ELISA);
6., in method of the present invention, after plasmid construction success, can preserve for a long time and be used for immune animal at any time.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Accompanying drawing explanation
Fig. 1 produces anti-human IL6 polyclonal antibody titre comparison diagram for using this method of the present invention and traditional immunization method;
Fig. 2 is the titre comparison diagram using this method of the present invention and traditional immunization method to produce anti-human hIgG3 polyclonal antibody;
Fig. 3 is that Western blot detects the anti-IL6 polyclonal antibody of rabbit producing also Isolation and purification with immunization of tatooing;
Fig. 4 is that Western blot detects with immunization production of tatooing, the anti-hIgG3 polyclonal antibody of rabbit of Isolation and purification.
Embodiment
Utilize the method for DNA plasmid immunizations Dispersal risk, the method comprises
1) antigen protein expression vector is built: the mode that the cDNA segment of goal gene is increased by reverse transcription PCR recalled from host cell gene group, be then cloned in expression vector;
2) plasmid DNA of purifying is prepared: utilize intestinal bacteria to prepare the plasmid DNA of purifying in a large number;
3) Plasmid DNA immunization: the mode of plasmid DNA being tatooed is injected in animal cuticle carries out expressing and immunity;
4) abstraction and purification antibody: adopt hybridoma screening monoclonal antibody separation and purification or collect serum and carry out polyclonal antibody extraction purification.
Particularly, step 1) middle employing pcDNA3.1 eukaryotic expression vector, goal gene is inserted between the NheI/EcorI double enzyme site on expression vector; The carrier built correctly is inserted in the protein expression reading frame on carrier with the exactness and sequence of guaranteeing goal gene sequence through sequence verification.
Particularly, step 2) be specially: the carrier built is transferred in DH5 α e. coli host cell and increases; Increased rear Isolation and purification plasmid.
Particularly, the present invention adopt Qiagen without intracellular toxin plasmid extraction kit Isolation and purification plasmid.
Particularly, described step 3) comprise the following steps:
S1: select animal back leg side to be position of tatooing, animal is anaesthetized, after entering narcosis for animal, electric shaver-for women removes the hair of need tatoo position and periphery, and cleans out;
S2: DNA plasmid good for purifying is applied in 1cm
2the position of the above-mentioned rejecting hair in scope, tatoos to it with syringe needle, complete after tatooing and coat pain relieving creme with clean cotton swab at skin surface of tatooing, and every two weeks repeats to tatoo immunity once;
S3: get blood between duration of immunity and be ELISA and Western blot and detect;
S4: until ELISA titre reach 1:8000 and be greater than negative control 3 standard deviations and Western blot detects and sees correct positive band time carry out fusion or the purified blood serum of splenocyte; Once reinforced immunological is carried out at the DNA plasmid of fusion or purified blood serum first 3 days muscle hemostasis purifying.
Particularly, step 4) in from serum the method for Isolation and purification antibody be in salting-out process, column chromatography purification, affinitive layer purification or ion exchange chromatography one or more combine.
It is below specific embodiment of the present invention, in the following embodiments, select Stealth Rotary to tatoo system, voltage of supply is set to need through suds cleaning before 4 volts of tattooing needles use and autoclave sterilization installs tattooing needle to tattooing apparatus, and adjustment depth of needle is 0.5mm; Syringe needle vibrational frequency is set to 100HZ.PcDNA3.1 derives from Invitrogen, Carlsbad, CA company; The reagent adopted in embodiment all can be bought by commercial channel and obtain.
Embodiment 1
Utilize DNA to tatoo immunity preparation rabbit anti-IL6 polyclonal antibody, concrete steps are as follows:
1) antigen protein expression vector is built: between the NheI/EcorI double enzyme site cDNA of goal gene IL6 being inserted into pcDNA3.1 eukaryotic expression vector; The carrier built correctly is inserted in the protein expression reading frame on carrier through sequence verification with the exactness and sequence of guaranteeing goal gene sequence;
2) plasmid DNA of purifying is prepared: be transferred to by the carrier built in DH5 α e. coli host cell and increase; Increased rear Isolation and purification plasmid;
3) Plasmid DNA immunization:
S1: select New Zealand white rabbit back leg side to be position of tatooing, adopts ketamine (ketamine; 90mg/kg) with xylazine (xylazine; Mixture 5mg/kg) is anaesthetized animal, enters after narcosis until New Zealand white rabbit, and electric shaver-for women removes the hair of need tatoo position and periphery, and cleans out;
S2: by step 2) to be diluted to concentration be 0.5mg/mL for DNA plasmid that purifying is good; Be applied in 1cm
2the position of the above-mentioned rejecting hair in scope, tatoos to it with syringe needle, complete after tatooing and coat pain relieving creme with clean cotton swab at skin surface of tatooing, and every two weeks repeats to tatoo immunity once, at every turn immune 100 μ L;
S3: the different time points (2,3,4,5 weeks) after immunity takes blood sample, and separation of serum does enzyme immunoassay (ELISA) and Western blot detects;
S4: until ELISA titre reach 1:8000 and be greater than negative control 3 standard deviations and Western blot detects and sees correct positive band time carry out the abstraction and purification of serum; DNA plasmid 100 microgram of 3 days muscle hemostasis purifying carries out primary reinforcement immunity before this step;
4) abstraction and purification antibody: collect serum and adopt column chromatography purification to carry out antibody extraction and purifying, obtain the anti-IL6 polyclonal antibody of rabbit.
Comparative example 1
According to the conventional method subcutaneous injection 200 microgram IL6 recombinant protein is carried out to New Zealand white rabbit, carry out immunity, every two weeks repeat immunity once, different time points (2,3,4,5 weeks) after immunity takes blood sample, and separation of serum does enzyme immunoassay (ELISA) and Western blot detects.The time identical with embodiment carries out collection and the antibody purification of serum; Once reinforced immunological is carried out at the DNA plasmid of purified blood serum first 3 days muscle hemostasis purifying; Collecting serum adopts column chromatography purification to carry out antibody extraction and purifying, obtains the anti-IL6 of rabbit.
Rabbit anti-IL6 albumen titre (mlU/ml) that embodiment 1 and comparative example 1 obtain is see Fig. 1.The result display of Fig. 1: be 100 times of comparative example through tatoo antibody titers that immunization produces of DNA.
Embodiment 2
Utilize DNA tatoo immunity preparation rabbit human immunoglobulins (hIgG3), concrete steps are as follows:
1) antigen protein expression vector is built: between the NheI/EcorI double enzyme site cDNA of goal gene hIgG3 being inserted into pcDNA3.1 eukaryotic expression vector; The carrier built correctly is inserted in the protein expression reading frame on carrier through sequence verification with the exactness and sequence of guaranteeing goal gene sequence;
2) plasmid DNA of purifying is prepared: be transferred to by the carrier built in DH5 α e. coli host cell and increase; Increased rear Isolation and purification plasmid;
3) Plasmid DNA immunization:
S1: select New Zealand white rabbit back leg side to be position of tatooing, adopts ketamine (ketamine; 90mg/kg) with xylazine (xylazine; Mixture 5mg/kg) is anaesthetized animal, enters after narcosis until New Zealand white rabbit, and electric shaver-for women removes the hair of need tatoo position and periphery, and cleans out;
S2: by step 2) to be diluted to concentration be 0.5mg/mL for DNA plasmid that purifying is good; Be applied in 1cm
2the position of the above-mentioned rejecting hair in scope, tatoos to it with syringe needle, complete after tatooing and coat pain relieving creme with clean cotton swab at skin surface of tatooing, and every two weeks repeats to tatoo immunity once, at every turn immune 200 μ L;
S3: the different time points (2,3,4,5 weeks) after immunity takes blood sample, and separation of serum does enzyme immunoassay (ELISA) and Western blot detects;
S4: until ELISA titre reach 1:8000 and be greater than negative control 3 standard deviations and Western blot detects and sees correct positive band time carry out collection and the antibody purification of serum; The DNA plasmid 100 μ g of 3 days muscle hemostasis purifying carries out primary reinforcement immunity before this step;
4) abstraction and purification antibody: collect serum and adopt column chromatography purification to carry out antibody extraction and purifying, obtain the anti-human hIgG3 polyclonal antibody of rabbit.
Comparative example 2
According to the conventional method subcutaneous injection 200 microgram hIgG3 recombinant protein is carried out to New Zealand white rabbit, carry out immunity, every two weeks repeat immunity once, different time points (2,3,4,5 weeks) after immunity takes blood sample, and separation of serum does enzyme immunoassay (ELISA) and Western blot detects.The time identical with embodiment carries out collection and the antibody purification of serum; Once reinforced immunological is carried out at the DNA plasmid of purified blood serum first 3 days muscle hemostasis purifying; Collecting serum adopts column chromatography purification to carry out antibody extraction and purified rabbit anti-human hIgG3 polyclonal antibody.
Rabbit anti-human hIgG3 albumen titre (mlU/ml) that embodiment 2 and comparative example 2 obtain is see Fig. 2.The result display of Fig. 2: be 100 times of comparative example through tatoo antibody titers that immunization produces of DNA.
The rabbit anti-IL6 anti-IL6 of polyclonal antibody rabbit (1:4000) and the anti-hIgG3 of rabbit (1:4000) polyclonal antibody Western blot detected result respectively see Fig. 3 and Fig. 4, Western blot result display DNA tatoo immunity preparation the anti-IL6 of rabbit and the anti-hIgG3 polyclonal antibody of rabbit have height specificity and avidity.
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (7)
1. utilize DNA to tatoo the method for passing immunization, it is characterized in that, the method comprises
1) antigen protein expression vector is built: the mode that the cDNA segment of goal gene is increased by reverse transcription PCR recalled from host cell gene group, be then cloned in expression vector;
2) plasmid DNA of purifying is prepared: utilize intestinal bacteria to prepare the plasmid DNA of purifying in a large number;
3) Plasmid DNA immunization: plasmid DNA is injected in animal cuticle in the mode of tatooing and carries out expressing and immunity;
4) abstraction and purification antibody: adopt hybridoma screening monoclonal antibody separation and purification or collect serum and carry out polyclonal antibody extraction purification.
2. method according to claim 1, is characterized in that, step 1) middle employing pcDNA3.1 eukaryotic expression vector, goal gene is inserted between the NheI/EcorI double enzyme site on expression vector; The carrier built correctly is inserted in the protein expression reading frame on carrier with the exactness and sequence of guaranteeing goal gene sequence through sequence verification.
3. method according to claim 1, is characterized in that, step 2) be specially: the carrier built is transferred in DH5 α e. coli host cell and increases; Increased rear Isolation and purification plasmid.
4. method according to claim 3, is characterized in that, adopt Qiagen without intracellular toxin plasmid extraction kit Isolation and purification plasmid.
5. method according to claim 1, is characterized in that, step 3) comprise the following steps:
S1: select animal back leg side to be position of tatooing, animal is anaesthetized, enters after narcosis until animal, electric shaver-for women removes the hair of need tatoo position and periphery, and cleans out;
S2: DNA plasmid good for purifying is applied in 1cm
2the position of the above-mentioned rejecting hair in scope, tatoos to it with syringe needle, complete after tatooing and coat pain relieving creme with clean cotton swab at skin surface of tatooing, and every two weeks repeats to tatoo immunity once;
S3: get blood between duration of immunity and be ELISA and Western blot and detect;
S4: until ELISA titre reach more than 1:8000 and be greater than negative control 3 standard deviations and Westernblot detects and sees correct positive band time carry out fusion or the purified blood serum of splenocyte; Once reinforced immunological is carried out at the DNA plasmid of fusion or purified blood serum first 3 days muscle hemostasis purifying.
6. method according to claim 5, is characterized in that, described animal is selected from mouse or New Zealand white rabbit.
7. method according to claim 1, is characterized in that, step 4) in from serum the method for Isolation and purification antibody be in salting-out process, column chromatography purification, affinitive layer purification or ion exchange chromatography one or more combine.
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| CN201410857000.3A CN104479016A (en) | 2014-12-31 | 2014-12-31 | Method for preparing antibodies through DNA tattooing immunization |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102257003A (en) * | 2008-12-19 | 2011-11-23 | 埃博灵克斯股份有限公司 | Method for generation of immunoglobulin sequences |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102257003A (en) * | 2008-12-19 | 2011-11-23 | 埃博灵克斯股份有限公司 | Method for generation of immunoglobulin sequences |
Non-Patent Citations (2)
| Title |
|---|
| QUAAK SGL等: "DNA tattoo vaccination:effect on plasmid purity and transfection efficiency of different topoisoforms", 《JOURNAL OF CONTROLLED RELEASE》 * |
| 曾伟伟等: "纹身法免疫增强DNA疫苗免疫效果的评价", 《畜牧兽医学报》 * |
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Application publication date: 20150401 |