CN1634992A - Method for optimizing and extracting rabbit-anti-human ATP7Bn33-629 polyclone antibodies - Google Patents

Abstract

This invention relates to a multi-clone antibody optimization and extraction method for human ATP7Bn33-629 by rabbit antibody and to a method for early diagnoses diseases by use of genes. The optimization method in this invention aims to the function area of ATP7B protein and selects amino acid protein with N end 33 to 629, which comprises six copper combination bits of N end and four important transmembrane function areas. This invention also provides a process method on base of the optimization, which forms GST gene fusion protein with ATP7B N end 33 to 629 amino acid aim sections to process the antibody according to the principles of initiating design.

Description

Preferred and the extraction method of the anti-people ATP7Bn33-629 of rabbit polyclonal antibody

Technical field

The present invention relates to the method that early gene diagnoses the illness, specifically to the preferred and extraction method of the anti-people ATP7Bn33-629 of the rabbit of hepatolenticular degeneration polyclonal antibody.

Background technology

Hepatolenticular degeneration claims Wilson disease (WD) again, is a kind of more common autosomal recessive hereditary diseases.Disease-causing gene is the ATP7B gene, coding ATP7B albumen.Its morbidity causes the decline of ATP7B protein function relevant with the ATP7B transgenation, but concrete pathogenesis is unclear so far.Because transgenation causes protein function to change, the binding ability of metallic copper and copper-protein precursor is descended, causing copper to be secreted into biliary amount from liver through bile duct reduces, and accumulate in a large number in liver, kidney, brain and cornea, occur corresponding symptom clinically, show as hepatorenal damage, extrapyramidal system pathology, disturbance of intelligence and Kayser-Fleischer ring etc.Although drive the copper treatment this disease there is certain curative effect, depends on early diagnosis and therapy.Because this sick early symptom is not true to type usually and shows variation, very easily by mistaken diagnosis and delay treatment so disability rate and case fatality rate are all very high, brings white elephant to patient family and society.Therefore, only carry out the research of ATP7B protein function, thereby disclose the WD pathogenesis, could fundamentally solve the early diagnosis and therapy problem of WD.

Research proteic function of ATP7B and mechanism of causing a disease thereof, high-quality ATP7B antibody is essential condition.Discover, the ATP7B assignment of genes gene mapping is in 13q14.3,1466 the amino acid whose ATP7B albumen of encoding, it is a kind of membranin, participates in the metabolic process of copper transmembrane transport, and its N-end has 6 copper binding sites, contain 8 and stride the film district, Transshipment Permitted copper permeate through cell membranes, therefore, the N-end is this proteic major function district.Because ATP7B gene coding region cDNA reaches 4398bp, it is very difficult to prepare the ATP7B antibody of containing full length coding region, contain several or amino acid whose antibody of dozens of or C end antibody so the ATP7B antibody of foreign scholar's preparation at present is the N end, do not comprise the major function district.Though the susceptibility of this antibody-like is higher, the specificity of antibody is not enough, has limited the degree of depth and range that antibody uses, as only being used for immunoblotting or only can being used for immunohistochemical methods, and causes false positive results easily, is not best ATP7B antibody therefore.

Summary of the invention

The objective of the invention is in the ATP7B gene, to optimize one section encoding sequence, and prepare the extracting method of polyclonal antibody.

The technical solution adopted for the present invention to solve the technical problems is: preparation antibody, and at first must the synthetic albumen for preparing antibody.The albumen of selecting for use which section albumen to prepare antibody and how synthesizing correct sequence is key of the present invention.Because the ATP7B gene reaches 4398bp, the ATP7B albumen that prepare full length coding region is very difficult, and is also unnecessary; If but the amino acid whose albumen of only synthetic N end dozens of prepares antibody, may cause antibodies specific not high enough.The anti-people ATP7Bn33-629 of a kind of rabbit polyclonal antibody preferred, it is characterized in that: at the proteic functional zone of ATP7B, the selected albumen of containing N end 33 to 629 amino acids prepares antibody, has comprised N 6 copper binding sites of end and 4 important film districts of striding.

Directly polypeptide synthesizes so big fragment and the correct albumen of encoding is very expensive by synthesizing, and also be unpractical, so the present invention selects the method for gst gene fusion rotein to prepare this target protein fragment.Described gst gene fusion rotein must make up and contains the segmental gst gene fusion vector of ATP7B gene N end 33 to 629 amino acids (1791bp) purposes; Design primer voluntarily according to the design of primers principle.

The concrete extracting method of the anti-people ATP7Bn33-629 of rabbit of the present invention polyclonal antibody:

1, preparation normal people full length coding region cDNA fragment: fresh liver is tissue-derived in acute liver trauma surgery patient, organizes once exsomatizing, and places in the liquid nitrogen rapidly; Take by weighing the 50mg liver organization, add 1mlTRIZOL liquid, homogenizer is mashed tissue, is transferred to behind the mixing in Eppendorf (Ep) pipe of 1.5ml.Add 150～250 μ l chloroforms, the whipping up and down of exerting oneself makes the abundant mixing of TRIZOL and chloroform, ice bath 5～10 minutes, and 4～10 ℃ 10000～13000 is centrifugal 10～30 minutes; The careful supernatant of drawing moves in the new 1.5ml Ep pipe, adds 450～550 μ l Virahols, and the Ep that overturns gently pipe 5～15 times was put ice bath 5～15 minutes, 4～10 ℃ 10000～13000 centrifugal 5～15 minutes, see pipe end adularescent precipitation; Remove supernatant, in precipitation, add 0.2～1ml, 70～75% ethanol, vibration, centrifugal 3～8 minutes of 4～10 ℃ of 7000～10000g; Remove supernatant, upset Ep pipe, drying at room temperature precipitation 15～30 minutes.-80 ℃ of preservations are standby behind the dissolution precipitation.This RNA is become cDNA with reverse transcription test kit (Invitrogen company) reverse transcription, be normal people's full length coding region cDNA fragment.

2, the selection of design of primers and gst gene fusion vector: the pcr amplification product fragment (target gene fragment) that guarantee to reach 1791bp is correct, and primer design is most important.For after guaranteeing target gene fragment (ATP7B gene N end 33-629 amino acid fragment) and the gst gene fusion vector being connected, frameshit does not take place and changes in the amino acid coding, in numerous gst gene fusion vectors, we have selected the pGEX4T-1 carrier, and select for use restriction enzyme SalI and NotI as tie point.Why selecting this 2 kinds of restriction endonucleases, be because do not have this 2 point of contacts on the ATP7B gene N end 33-629 amino acid fragment, and they also is more common restriction enzyme.Design of primers adopts primer-design software usually, but herein, we are without primer-design software, design primer voluntarily according to the design of primers principle, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series.Designed 6 primers altogether, sequence is as follows:

1R 5 '-gTCgACCAATGAAGAAGAGT-3 ' (containing the SalI point of contact)

1F 5 '-gCggCCg_ cATgAAAgCCAAT-3 ' (containing the NotI point of contact)

2R?5’-gAAAggCATCATCAgCATgAAgg-3’

3F?5’-gCAggTCATgCCCTCCACCCg-3’

4R?5’-gggACCAATTgATATTgAgCg-3’

5F?5’-gCCTTTCCTgCCATCAAgg-3’

1R and 1F are used for the pcr amplification target gene fragment, i.e. ATP7B gene N end 33-629 amino acid fragment, 2R, 3F, 4R and 5F are used for checking order and confirm the target gene fragment sequence that increased.

3, prepare target gene fragment and being connected on the gst gene fusion vector: with normal people's full length coding region cDNA fragment is template, adopts primer 1R, 1F and high-fidelity pcr amplification test kit amplification ATP7B gene N end 97～1887cDNA sequence.Use TA clone test kit the PCR product is connected on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium and adopt plasmid extraction purification kit extracting and purifying plasmid.SalI and NotI double digestion confirm positive TA clone and adopt primer 2 R, 3F, 4R and 5F and ABIPRISM ^RThe exactness that is connected the target gene fragment sequence on the PCR2.1 carrier is identified in the order-checking of 3700 dna sequence analysis instrument.Altogether picking 56 positive TA clones just chosen a right-on clone of sequence.Use the correct TA clone of SalI and NotI double digestion, reclaim target gene fragment and be connected to and go up and transform through the gst gene fusion vector (pGEX4T-1) of SalI and the recovery of NotI double digestion.

4, the expression that contains the gst gene fusion vector of target gene fragment is identified: 1 of picking transforms good gst gene fusion vector clone, shake bacterium with 5ml 2YT substratum, centrifugal collection bacterium and preparation gst gene fusion rotein in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, the blue dyeing of coomassie, the result shows that gst gene fusion rotein clip size is correct, expresses good.

5, preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 4～15 ℃ of enzymes of application Thrombin are cut and are spent the night, cross post in second day and collect the ATP7Bn33-629 target protein, adopt PAGE-SDS glue further to confirm the fragment and the purity of target protein and detect protein concentration.

6, preparation and the anti-people ATP7Bn33-629 of purified rabbit polyclonal antibody (Rabbit anti-humanATP7Bn33-629 polyclonal antibody): subcutaneous multi-point injection immunizing rabbit after ATP7Bn33-629 target protein and the adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of ATP7Bn33-629 polyclonal antibody, and be 1: 2000 this moment.After this booster immunization was 1 time in per 7～14 days, all got blood 1ml from auricular vein at every turn, and the ELISA method detects antibody titers.Behind the booster immunization 3 times, the titre that the ELISA method detects antibody has reached 1: 16000.Adopt the specificity of western blotting method checking antibody, the result confirms that this antibody has high degree of specificity, nothing but specific band.The above results points out the susceptibility of this antibody and specificity all very high.At this moment, from the rabbit heart puncturing extracting blood, Immobilized ProteinAColumn (Pierce company) antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-20 ℃.We are with the anti-people ATP7Bn33-629 of this antibody called after rabbit polyclonal antibody (Rabbitanti-human ATP7Bn33-629 polyclonal antibody).

7, the application of the anti-people ATP7Bn33-629 of rabbit polyclonal antibody:

(1) immunoblotting: 2.5～10% skimmed milks are as confining liquid, and room temperature closed protein electricity changes 30～60 fens kinds of film; The anti-people ATP7Bn33-629 of rabbit antibody (concentration is 5 μ g/ μ l) is anti-as one, dilution in 1: 500～1: 1000, and room temperature is shaken to educate to shake to educate in 2～6 hours or 4～8 ℃ and is spent the night, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000～1: 10000, and room temperature is shaken and is educated 45～90 minutes, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds, the colour developing of horseradish peroxidase substrate, compressing tablet and punching.The results are shown in Figure 1.

(2) immunofluorescence dyeing: ATP7B expression plasmid transfection CHO cell, 4～8% Paraformaldehyde 96 fixed cells, 5～10% lowlenthal serum room temperature closing cells 30～60 minutes; The anti-people ATP7Bn33-629 of rabbit antibody (concentration is 5 μ g/ μ l) dilution in 1: 50～1: 150, room temperature reaction 1～3 hour, 1XPBS shake washes 3 times, each 5～10 minutes kinds; The goat anti-rabbit igg of fluorescence is anti-as two redly, dilution in 1: 100～1: 300, and room temperature reaction 30～60 minutes, 1XPBS shake washes 3 times, each 5～10 minutes; The mounting mirror is observed down, the results are shown in Figure 2.

The invention has the beneficial effects as follows: because the present invention design and prepared the anti-people ATP7Bn33-629 of rabbit polyclonal antibody, nearly 600 amino acid that contain the ATP7B albumen n end, its main functional zone have been contained, be 6 copper binding sites and 4 important film districts of striding of N end, thereby guaranteed the high degree of specificity of antibody, helped to carry out the research of ATP7B protein function and the early stage rapid gene diagnosis of WD.The present invention has simultaneously proposed the extracting method of its preparation on base of optimum selection, so for the early gene diagnosis of hepatolenticular degeneration extremely long-pending effect is arranged.

Description of drawings:

Fig. 1 is the photo as a result that antibody of the present invention is used for immunoblotting.Mark 1 is not for expressing the proteic sample of ATP7B; 3 and 4 samples for the expression of ATP7B protein positive.Clip size is 163Kd, between 97Kd and 220Kd.

Fig. 2 is the alternate growth mirror of cell figure below of ATP7B expression plasmid on transfectional cell of the present invention (fluorescence redly) and the untransfected.

Embodiment

Embodiment 1:

At the proteic functional zone of ATP7B, the selected albumen of containing N end 33 to 629 amino acids prepares antibody, has comprised 6 copper binding sites and 4 important film districts of striding of N end.

Embodiment 2:

Select the method for gst gene fusion rotein to prepare this target protein fragment, the gst gene fusion rotein must make up and contains ATP7B gene N and hold the segmental gst gene fusion vector of 33 to 629 amino acids (1791bp) purposes; Design primer voluntarily according to the design of primers principle.

Concrete extracting method:

1, preparation normal people full length coding region cDNA fragment: fresh liver is tissue-derived in acute liver trauma surgery patient, organizes once exsomatizing, and places in the liquid nitrogen rapidly; Take by weighing the 50mg liver organization, add 1ml TRIZOL liquid, homogenizer is mashed tissue, is transferred to behind the mixing in Eppendorf (Ep) pipe of 1.5ml.Add 200 μ l chloroforms, firmly up and down whipping makes the abundant mixing of TRIZOL and chloroform, ice bath 5 minutes, centrifugal 15 minutes of 4 ℃ of 12000g; The careful supernatant of drawing moves in the new 1.5ml Ep pipe, adds 500 μ l Virahols, and the Ep that overturns gently pipe 5 times was put ice bath 10 minutes, and centrifugal 10 minutes of 4 ℃ of 12000g see pipe end adularescent precipitation; Remove supernatant, in precipitation, add 1ml 75% ethanol, vibration, centrifugal 5 minutes of 4 ℃ of 7500g; Remove supernatant, upset Ep pipe, drying at room temperature precipitation 20 minutes ,-80 ℃ of preservations are standby behind the dissolution precipitation.This RNA is become cDNA with reverse transcription test kit (Invitrogen company) reverse transcription, be normal people's full length coding region cDNA fragment.

2, the selection of design of primers and gst gene fusion vector: for after guaranteeing target gene fragment (ATP7B gene N end 33-629 amino acid fragment) and the gst gene fusion vector being connected, frameshit does not take place and changes in the amino acid coding, in numerous gst gene fusion vectors, selected the pGEX4T-1 carrier, and select for use restriction enzyme SalI and NotI as tie point, because do not have this 2 point of contacts on the ATP7B gene N end 33-629 amino acid fragment, and they also are more common restriction enzymes.Design of primers adopts primer-design software usually, but herein, without primer-design software, design primer voluntarily according to the design of primers principle, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series.Designed 6 primers altogether, sequence is as follows:

1R 5 '-gTCgACCAATGAAGAAGAGT-3 ' (containing the SalI point of contact)

1F 5 '-gCggCCg_ cCATgAAAgCCAAT-3 ' (containing the NotI point of contact)

2R?5’-gAAAggCATCATCAgCATgAAgg-3’

3F?5’-gCAggTCATgCCCTCCACCCg-3’

4R?5’-gggACCAATTgATATTgAgCg-3’

5F 5 '-gCCTTTCCTgCCATCAAgg-3 ' 1R and 1F are used for the pcr amplification target gene fragment, i.e. ATP7B gene N end 33-629 amino acid fragment, 2R, 3F, 4R and 5F are used for checking order and confirm the target gene fragment sequence that increased.For 1F and 1R design of primers certain skill is arranged, 5 ' end at 1F has added SalI point of contact sequence gTCgAC, thereafter base sequence is consistent with ATP7B gene N end 33-36 amino acid fragment, 1R is a reverse sequence, added NotI point of contact sequence gCggCCgC at its 5 ' end, thereafter base sequence is consistent with ATP7B gene N end 625-629 amino acid fragment, and target gene fragment can be linked on the pGEX4T-1 carrier by SalI and NotI double digestion.What deserves to be mentioned is that all with A ending, rather than conventional with G or C ending at 3 ' end of 1F and 1R primer, purpose is the easier positive colony of choosing when being the TA clone.2R, 3F, 4R and 5F design of primers are deferred to conventional design of primers principle and are got final product.

3, prepare target gene fragment and being connected on the gst gene fusion vector: with normal people's full length coding region cDNA fragment is template, adopts primer 1R, 1F and high-fidelity pcr amplification test kit amplification ATP7B gene N end 97-1887cDNA sequence.Use TA clone test kit the PCR product is connected on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium and adopt plasmid extraction purification kit extracting and purifying plasmid.SalI and NotI double digestion confirm positive TA clone and adopt primer 2 R, 3F, 4R and 5F and ABIPRISM ^RThe exactness that is connected the target gene fragment sequence on the PCR2.1 carrier is identified in the order-checking of 3700 dna sequence analysis instrument.Altogether picking 56 positive TA clones chosen a right-on clone of sequence.Use the correct TA clone of SalI and NotI double digestion, reclaim target gene fragment and be connected to and go up and transform through the gst gene fusion vector (pGEX4T-1) of SalI and the recovery of NotI double digestion.

4, the expression that contains the gst gene fusion vector of target gene fragment is identified: 1 of picking transforms good gst gene fusion vector clone, shake bacterium with 5ml 2YT substratum, centrifugal collection bacterium and preparation gst gene fusion rotein in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, the blue dyeing of coomassie, the result shows that gst gene fusion rotein clip size is correct, expresses good.

5, preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 4～15 ℃ of enzymes of application Thrombin are cut and are spent the night, cross post in second day and collect the ATP7Bn33-629 target protein, adopt PAGE-SDS glue further to confirm the fragment and the purity of target protein and detect protein concentration.

6, preparation and the anti-people ATP7Bn33-629 of purified rabbit polyclonal antibody (Rabbit anti-humanATP7Bn33-629 polyclonal antibody): subcutaneous multi-point injection immunizing rabbit after ATP7Bn33-629 target protein and the adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of ATP7Bn33-629 polyclonal antibody, and be 1: 2000 this moment.After this booster immunization was 1 time in per 7～14 days, all got blood 1ml from auricular vein at every turn, and the ELISA method detects antibody titers.Behind the booster immunization 3 times, the titre that the ELISA method detects antibody has reached 1: 16000.Adopt the specificity of western blotting method checking antibody, the result confirms that this antibody has high degree of specificity, nothing but specific band.At this moment, from the rabbit heart puncturing extracting blood, ImmobilizedProteinAColumn (Pierce company) antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-20 ℃.

7, the application of the anti-people ATP7Bn33-629 of rabbit polyclonal antibody:

(1) immunoblotting (seeing also Fig. 1): 2.5～10% skimmed milks are as confining liquid, and room temperature closed protein electricity changes 30～60 fens kinds of film; The anti-people ATP7Bn33-629 of rabbit antibody (concentration is 5 μ g/ μ l) is anti-as one, dilution in 1: 500～1: 1000, and room temperature is shaken to educate to shake to educate in 2～6 hours or 4～8 ℃ and is spent the night, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000～1: 10000, and room temperature is shaken and is educated 45～90 minutes, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds, the colour developing of horseradish peroxidase substrate, compressing tablet and punching.

(2) immunofluorescence dyeing (seeing also Fig. 2): ATP7B expression plasmid transfection CHO cell, 4～8% Paraformaldehyde 96 fixed cells, 5～10% lowlenthal serum room temperature closing cells 30～60 minutes; The anti-people ATP7Bn33-629 of rabbit antibody (concentration is 5 μ g/ μ l) dilution in 1: 50～1: 150, room temperature reaction 1～3 hour, 1XPBS shake washes 3 times, each 5～10 minutes kinds; The goat anti-rabbit igg of fluorescence is anti-as two redly, dilution in 1: 100～1: 300, and room temperature reaction 30～60 minutes, 1XPBS shake washes 3 times, each 5～10 minutes; The mounting mirror is observed down.

Claims (4)

1, the anti-people ATP7Bn33-629 of a kind of rabbit polyclone body is preferred, it is characterized in that: at the proteic functional zone of ATP7B, the selected albumen of containing N end 33 to 629 amino acids prepares antibody, has comprised 6 copper binding sites and 4 important film districts of striding of N end.

2, the anti-people ATP7Bn33-629 of a kind of rabbit polyclone body is preferred, it is characterized in that: select the method for gst gene fusion rotein to prepare this target protein fragment, described gst gene fusion rotein must make up and contains the segmental gst gene fusion vector of ATP7B gene N end 33 to 629 amino acids (1791bp) purposes; Design primer voluntarily according to the design of primers principle.

3, the concrete extracting method of the anti-people ATP7Bn33-629 of rabbit of the present invention polyclonal antibody:

(1) preparation normal people full length coding region cDNA fragment: fresh liver is tissue-derived in acute liver trauma surgery patient, organizes once exsomatizing, and places in the liquid nitrogen rapidly; Take by weighing the 50mg liver organization, add 1mlTRIZOL liquid, homogenizer is mashed tissue, is transferred to behind the mixing in Eppendorf (Ep) pipe of 1.5ml.Add 150～250 μ l chloroforms, whipping makes the abundant mixing of TRIZOL and chloroform up and down, ice bath 5～10 minutes, and 4～10 ℃ 10000～13000 is centrifugal 10～30 minutes; Draw supernatant and move in the new 1.5ml Ep pipe, add 450～550 μ l Virahols, the Ep that overturns gently pipe 5～15 times was put ice bath 5～15 minutes, 4～10 ℃ 10000～13000 centrifugal 5～15 minutes, see pipe end adularescent precipitation; Remove supernatant, in precipitation, add 0.2～1ml70～75% ethanol, vibration, centrifugal 3～8 minutes of 4～10 ℃ of 7000～10000g; Remove supernatant, upset Ep pipe, drying at room temperature precipitation 15～30 minutes;-80 ℃ of preservations are standby behind the dissolution precipitation.This RNA is become cDNA with reverse transcription test kit reverse transcription, be normal people's full length coding region cDNA fragment;

(2) selection of design of primers and gst gene fusion vector: selected the pGEX4T-1 carrier, and selected for use restriction enzyme SalI and NotI as tie point; Design primer voluntarily according to the design of primers principle, accomplish that same primer do not have palindrome hairpin structure, 5 ' end is not complementary with 4 bases of 3 ' end, and forward and reverse primer sequence is not complementary, Tm value close (2A+2T+4G+4C), and on Genebank, can not find homologous series; Designed 6 primers altogether, sequence is as follows:

1R 5 '-gTCgACCAATGAAGAAGAGT-3 ' (containing the SalI point of contact)

1F 5 '-gCggCCg cATgAAAgCCAAT-3 ' (containing the NotI point of contact)

2R?5’-gAAAggCATCATCAgCATgAAgg-3’

3F?5’-gCAggTCATgCCCTCCACCCg-3’

4R?5’-gggACCAATTgATATTgAgCg-3’

5F?5’-gCCTTTCCTgCCATCAAgg-3’

1R and 1F are used for the pcr amplification target gene fragment, i.e. ATP7B gene N end 33-629 amino acid fragment, 2R, 3F, 4R and 5F are used for checking order and confirm the target gene fragment sequence that increased;

(3) preparation target gene fragment and being connected on the gst gene fusion vector: with normal people's full length coding region cDNA fragment is template, adopts primer 1R, 1F and high-fidelity pcr amplification test kit amplification ATP7B gene N end 97-1887cDNA sequence; Use TA clone test kit the PCR product is connected on the PCR2.1 carrier, transform and the positive TA clone of picking, shake bacterium and adopt plasmid extraction purification kit extracting and purifying plasmid; SalI and NotI double digestion confirm that positive TA clone and employing primer 2 R, 3F, 4R and 5F and the order-checking of ABIPRISMR3700 dna sequence analysis instrument identify the exactness that is connected the target gene fragment sequence on the PCR2.1 carrier; Use the correct TA clone of SalI and NotI double digestion, reclaim target gene fragment and be connected to and go up and transform through the gst gene fusion vector (pGEX4T-1) of SalI and the recovery of NotI double digestion;

(4) expression that contains the gst gene fusion vector of target gene fragment is identified: 1 of picking transforms good gst gene fusion vector clone, shake bacterium with 5ml 2YT substratum, centrifugal collection bacterium and preparation gst gene fusion rotein in a small amount, adopt PAGE-SDS gel electrophoresis protein isolate fragment, the blue dyeing of coomassie shows gst gene fusion rotein clip size;

(5) preparation and purifying gst gene fusion rotein and collection target protein: prepare the gst gene fusion rotein in a large number, crossing column purification reclaims also quantitatively, 4～15 ℃ of enzymes of application Thrombin are cut and are spent the night, cross post in second day and collect the ATP7Bn33-629 target protein, adopt PAGE-SDS glue further to confirm the fragment and the purity of target protein and detect protein concentration;

(6) subcutaneous multi-point injection immunizing rabbit after preparation and the anti-people ATP7Bn33-629 of purified rabbit polyclonal antibody ATP7Bn33-629 target protein and the adjuvant homogenate, get blood 1ml from the auricular vein of rabbit after 1 month, solidify back centrifuging and taking serum, the ELISA method detects the titre of ATP7Bn33-629 polyclonal antibody, and be 1: 2000 this moment; After this booster immunization was 1 time in per 7～14 days, all got blood 1ml from auricular vein at every turn, and the ELISA method detects antibody titers; Behind the booster immunization 3 times, the titre that the ELISA method detects antibody has reached 1: 16000; Adopt the specificity of western blotting method checking antibody, the result confirms that this antibody has high degree of specificity, nothing but specific band; From the rabbit heart puncturing extracting blood, ImmobilizedProteinAColumn (Pierce company) antibody purification is adopted in the packing of centrifugal collection antibody serum, and detectable level and packing are stored in-20 ℃.

4, the application of the anti-people ATP7Bn33-629 of rabbit polyclonal antibody is characterized in that:

(1) immunoblotting: 2.5～10% skimmed milks are as confining liquid, and room temperature closed protein electricity changes 30～60 fens kinds of film; The anti-people ATP7Bn33-629 of rabbit antibody (concentration is 5 μ g/ μ l) is anti-as one, dilution in 1: 500～1: 1000, and room temperature is shaken to educate to shake to educate in 2～6 hours or 4～8 ℃ and is spent the night, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds; The goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, dilution in 1: 5000～1: 10000, and room temperature is shaken and is educated 45～90 minutes, and the 1XTBST damping fluid shakes washes 3 times, each 5～15 minutes kinds, the colour developing of horseradish peroxidase substrate, compressing tablet and punching;

(2) immunofluorescence dyeing: ATP7B expression plasmid transfection CHO cell, 4～8% Paraformaldehyde 96 fixed cells, 5～10% lowlenthal serum room temperature closing cells 30～60 minutes; Its concentration of the anti-people ATP7Bn33-629 of rabbit antibody is 5 μ g/ μ l, dilution in 1: 50～1: 150, and room temperature reaction 1～3 hour, 1XPBS shake washes 3 times, each 5～10 minutes kinds; The goat anti-rabbit igg of fluorescence is anti-as two redly, dilution in 1: 100～1: 300, and room temperature reaction 30～60 minutes, 1XPBS shake washes 3 times, each 5～10 minutes; The mounting mirror is observed down.

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