CN1059928C - Method for preparing antigenic protein capable of testing C-type hepatitis by using colibacillus - Google Patents

Abstract

The present invention relates to a method. In the method, colon bacillus expression systems are used for expressing a section of the DNA fragments of the core antigen parts of hepatitis C viruses (HCV); proteins expressed through thermal induction can be specifically identified by blood serum of most of patients infected by non A and B type hepatitis but can not be identified by blood serum of healthy people; compared with the anti C100-3 diagnostic results through yapei reagents, the diagnostic results of hepatitis C obtained through the antigen proteins are more sensitive and can perfect each other for reducing false negative results; thus, the antigen proteins prepared by the present invention can be used as a good detecting reagent for detecting hepatitis C.

Description

Utilize the intestinal bacteria preparation to can be used to the method for the antigen protein of testing C-type hepatitis

The invention belongs to the method for preparing the testing C-type hepatitis antigen protein.

After the introduction of the 1970's was done the screening of hepatitis B virus to the blood of donations, infected B type hepatitis case just significantly reduced via route of transfusion.Yet, still have many hepatitis cases that infect via blood transfusion to be caused by virus.Causing the hepatitis of this class, is not that hepatitis A virus (Hepatitis A virus HAV) neither hepatitis B virus (HepatitisB virus usually; HBV), so be called non-a non-b hepatitis (NANBH).The caused hepatitis of this virus becomes very serious clinically problem at present, because it almost accounts for more than 90% of hepatitis case of all blood transfusion postoperative infections.And the infected most likely develops into chronic hepatitis, and then develops into liver cirrhosis or liver cancer.Cause the virus of this non-a non-b hepatitis, though the research of many people's effort for a long time, once very unclear, until U.S. Chiron company, (Seience 244 for people such as Choo; 359-362,1989) found that a single-stranded RNA virus may be after causing the main pathogen of this non-a non-b hepatitis, just the non-a non-b hepatitis that blood transfusion is infected has further understanding.Now this RNA viruses be called as hepatitis C virus (Hepatitis C virus, HCV) in fact HCV in the blood of infected individuals, exist very little, so up to the present all can't directly isolate virus or by microscopic examination to this virus.This also is the reason that why all always can't break through for a long time.People such as Choo utilize the blood that is got by patient directly to take away to infect chimpanzee, are made the gene pool of a gt 11 again by isolated viral RNA in the chimpanzee blood, utilize patient's serum to screen then.Through method thus, they find out the dna segment (5-1-1) of one section HCV, and the expressed antigen that comes out of this segment, though special association reaction can be arranged with the serum of hepatitis sufferer.This just begins to have had the research about HCV.Other little cDNA segments that they further will find successively link together becomes one section long cDNA, is referred to as C100-3.After expressing the C100-3 segment and become antigen protein with saccharomycetic expression vector again, just check the serum samples of large quantities of C type hepatitis sufferers that get by the U.S., Italy or Japan with enzyme immunoassay, found that the above blood of 70-80% sliding all with this antigen-reactive.Further confirm the caused by HCV really of most non-a non-b hepatitis.

Through a plurality of breadboard effort, the genome of HCV (genome) is all cloned out at present.It is that an about 10kb is long, and the RNA of underlying stock forms.Utilize sequence similarity character (sequence homology) to analyze, HCV be very similar to flavivirus (genus) (flavivirus) and pestivirus (genus) (pestivirus), but be not identical.This viral full-length gene group only can be synthesized the protein of about 3011 amino acid longs.Obviously this long protein molecule need pass through further cracking (cleavage) again and become big and small different virus protein.If words with the genome of flavivirus (genus), its formed virus protein mainly comprises (being risen by the N terminal number): core (nucleocapsid, C), membranin (membrane), overcoat albumen (envelope, E) and five Nonstructural Proteins (nonstructrural proteins, NS1-NS5); And as if the genome by pestivirus, except core protein (C), be exactly two overcoat albumen (E1 and E2 all are glucoprotein (glycoprotein)) then, nonstructural proteins then has only four (NS2-NS5).HCV then is very similar to the latter's structure, and promptly after core protein, two glycoprotein are arranged, and (E1 NS1/E2), is thereafter four Nonstructural Proteins (NS2-NS5); The structure that it is correct and institute's synthetic protein size and function etc. still have to be determined.

The blood sample of check Blood Center certainly will be one and prevent that HCV from causing the main policies of blood transfusion postoperative infection hepatitis.For this reason, Chiron company has been cloned in the HCV dna segment (C100-3) of its gained and has made a testing reagent in the yeast expressed carrier.In fact also has one section class superoxide dismutase (superoxide dismutase) gene (SOD) on this carrier.The segment of HCV and SOD form a fusion rotein (fusion protein), are combined by 154 amino acid of SOD and 363 amino acid of HCV.This C100-3 is that the montage of a non-structural protein white region (NS-3 of NS-4 and part) institute is got off, and so its sequence is all closely similar in order to check different HCV somatotypes quite effective on the country variant isolated HCV genome of institute (genome).But this testing reagent is also intact no all roses, because in fact, the measured HCV incidence of C100-3 also has only the C type hepatitis about 70-80%.Why can be like this? may be because NS-4 be a Nonstructural Protein, after the virus infection individuality, causing antibody occurs, resist these nonstructural proteins, the needed time is longer, perhaps just can occur after will reaching half a year, so when utilizing C100-3 to measure, might cause false-negative result.And, when patient blood presents than low reaction numerical value, be Abbott and the Ortho two tame products that utilize Chiron antigen to be made equally, the result of mutual contradiction often just appears.In addition, some has the people of autoimmunity (auto-immune) disease again, and its serum may have the antibody to SOD, so when detecting with this testing reagent, false resistive result must be arranged.

The objective of the invention is to provide a kind of preparation to can be used to the method for the antigen protein of testing C-type hepatitis virus (HCV), and with this antigen as the HCV testing reagent in order to compensate the shortcoming of present HCV testing reagent, assay more accurately is provided.

The present invention is directed to present understanding to HCV, and the shortcoming of present agents useful for same, having selected especially with structural protein is that main gene (refers to core protein at this, nucleocapsid), utilize coli expression carrier,, thereby give expression to a large amount of antigen proteins with the technology of recombination.And this antigen protein does not have any other extra, unnecessary sequence.

The present invention is at first from selected non-a non-b hepatitis patients'blood, isolate viral RNA, utilize Japanese document (Jpn.J.Exp.Med.60:167-177 again, 1990) the section H CV sequence of being delivered, with the synthetic a pair of introduction of chemical synthesis, the cDNA segment of synthesizing one section HCV with the method for RNA reverse transcription and polymerase chain reaction (RT-PCR) in advance.This segment has been forgiven 5 ' and has not been translated district (untranslated region), and core protein (core) zone.The cDNA segment that gets by PCR, and carry out the DNA sequencing work of total length, and obtain the sequence that this part belongs to the HCV isolate of Taiwan.Warp and U.S.'s reported sequence, or Japanese reported sequence is compared nearly 95% same originality.And then the sequence of coming out according to own sequencing, in the zone of the core protein of inferring, synthesize another to introduction, and after the sequence of its C end introduction, specially add a TAA for translating terminator (termination codon), utilize round pcr again, the segment of synthetic core protein gene, directly this segment DNA is grown then into transcribe in the λ of colibacillus expression vector P promoter (promoter, P).This promoter is very capable at e. coli expression.For avoiding its to express the growth that too early influence thalline, we with this carrier grow a strain contain cI 857 inhibition subbases because of the host in.These cI 857 genes can be made a kind of thermally sensitive inhibition-cI.Usually when 30 ℃ of-32 ℃ of culture condition, this suppresses the expression that son can suppress Promoter, but if when temperature risen to 42 ℃, and suppressing son promptly can sex change and lose the effect of inhibition.The P promoter is so can express its genes carried.Utilize this expression system, behind the Overheating Treatment several hrs, and obtain the cAg protein of capacity.Then bacterium is broken with mechanical means, get final product purifying, isolate this protein, with usefulness as testing reagent.

By the prepared core protein of intestinal bacteria, utilize policapram gel electrophoresis after separating, again with west ink marks method (west blot), carry out association reaction with C type hepatitis patient's serum, just can be used as the usefulness that detects C type hepatitis.Description of drawings:

Fig. 1: for by RT-PCR pulsating nucleic acid of prepared HCV cDNA and aminoacid sequence

The sequence of nucleic acid is to get according to the DNA sequencing among the figure, and total length is 584bp altogether, and the relative position of every capable last nucleic acid of sequence is represented on the right of figure.The special PCR reaction of being done during for the cAg part of the introduction sequence (A1 and A2) that RT-PCR when reaction is used and second step and the introduction sequence (C1 and C2) of usefulness rises with the grid frame respectively or represents with arrow.Contain one and translate terminator sequence (TAA) as 3 ' end introduction of the second step PCR reaction is then more so that protein is translated can finish and a Sal I (GTCGAC) sequence.The rotaring intertranslating start point of cAg (ATG) is begun by the 212nd nucleic acid position.

Fig. 2: for expressing the structure of plastid pG408N-C

The space part is represented the P promoter among the figure, is the dna segment of one section about 380bp.Dash area is then represented the cDNA segment (36bp) of HCV.The fine rule of garden circle is partly represented the plastid part of pGEM-4.

Fig. 3: be the expression analysis of HGV antigen protein in intestinal bacteria

Contain express physique escherichia coli host respectively through 30 ℃ of culture condition, or 42 ℃ of thermal treatment be incubated at again after 15 minutes 37 ℃ following 3 hours, then the centrifugal recovery of whole thalline with 2X cracking damping fluid dissolving, use 12.5% SDS-policapram glue with protein separation (seeing example 2 and 3) again.The To representative is induced without thermal treatment, and the Ta representative is induced through Overheating Treatment.M represents the mark of known protein matter size.The position of the HCV cAg that the arrow indication is.

Fig. 4: carry out west ink marks method analysis for utilizing chronic hepatitis c patient serum

Method described in wherein similar Fig. 3, bacterium protein are after separating through running gel, and promptly the serum with four patients (1-4) and a normal people (5) carries out west ink marks method analysis (seeing example 3).The arrow indication is then represented the protein of expressing through thermal induction, and it can be patients serum institute recognition, but in (N) normal people's serum, this protein of newly expressing then can not be identified.The pulsating clone of the proteinic cDNA of example 1:HCV cAg

Be the cDNA of screening Taiwan district HCV virus, the partial sequence (Jpn.J.Exp.Med.60:167-177,1990) according to the Japanese virus strain of delivering recently makes two introduction A1 and A2 (see figure 1) respectively.And utilize this to introduction, and be material with the virus in hospital's sufferer blood, carry out RT-PCR.

At first the patients serum with 100 μ l places test tube, and adds denaturing soln (the 4M thiocyanic acid sodium salt of 500 μ l; 25mM Trisodium Citrate pH7.0; 0.5% Sarcosyl; 0.1M beta-mercaptoethanol) mix, add 60 μ l 2M Trisodium Citrates (PH4.0) successively, 600 μ l are with water saturated phenol, and the chloroform of 120 μ l: primary isoamyl alcohol (49: 1), after acutely mixing 30 seconds, left standstill 15 minutes on ice.Then with 10000rpm.4 ℃ centrifugal 10 minutes down, take out upper strata liquid to another in vitro newly, add the glycogen of isopyknic Virahol and 10 μ g after, mix, in-20 ℃ of standing over night (or-70 ℃, 1 hour) with precipitated rna.Again with 12000rpm.4 ℃, centrifugal 20 minutes, supernatant liquor is removed, behind the salinity in the 75% alcohol flush away RNA precipitation, remove remaining alcohol with vacuum drier.RNA is dissolved in right amount in the water of (about 5 μ l), and prepares to carry out RT-PCR.

The RT-PCR the first step is carried out reverse transcription reaction earlier.Aforementioned RNA is added water to 6.5 μ l, boil 5 minutes with boiling water after, place frozen water cooling rapidly, centrifugal slightly after, add reaction reagent: 10X PCR damping fluid 2 μ l; 10mM dATP 2 μ l; 10mM dTTP 2 μ l; 10mM dCTP2 μ l; 10mM dGTP 2 μ l; Random hecamer (100ng/ μ l) 2 μ l; RNasin (40u/ μ l) 0.5 μ l 2 μ l; MuL V RT (200u/ μ l) 1 μ l; [10X PCR damping fluid composition is as follows: 500mM KCl, 100mM Tris-HCl pH8.3,15mM MgCl ₂, 0.1% (W/V) gelatin (can omit)] mix, reacted 1 hour down in 37 ℃.Then, place cooled on ice standby rapidly, in the test tube of PCR special use, add following reagent then: 38 μ l H sample heating 5 minutes ₂O, 4.5 μ l 10XPCR damping fluids, each 1 μ l (100ng/ul) of A1 and A2 introduction, 0.5 μ l Tag archaeal dna polymerase (5u/ μ l), and the above-mentioned reverse transcription reaction product of 5 μ l.After all reagent mix were even, the mineral oil that adds 50 μ l at liquid level placed in the thermal cycler instrument (Perkin Elmer Cetus) with being about to test tube, set reaction conditions and was: 94 ℃, and 30 seconds; 60 ℃, 30 seconds; And 74 ℃, 90 seconds is a circulation.

Through getting the cDNA segment of 0.587kb in a large number after 35 circulations.Then this cDNA segment is cloned into the pGem-4Z carrier, gets the plastid of a pHCV C-4 and then utilize the method for Sanger to carry out DNA sequencing (see figure 1).It is position (ATG) beginning by the 212nd nucleic acid that the protein that we know HCV by the sequence among Fig. 1 is translated.If compare with the sequence of flavivirus (genus), core protein is at the N of whole length protein end.And infer that by document cAg approximately contains amino acid (J.Gen, Virol, 71 about 190; 3027-3033,1990), thus our the cDNA segment that makes only contain 119 aminoacid sequences of its N end.

In order to utilize intestinal bacteria to express this section core protein gene, designed another again to introduction C1 and C2 (Fig. 1), be template with pHCV C-4, will belong to the dna sequence dna of core protein scope, go again with the method for PCR and increase out.Used template DNA is 1ng in the PCR reaction, C1 and C2 introduction respectively are 1 μ g, add the 10X PCR reaction soln of 10 μ l and four kinds of nucleic acids (dATP, dGTP of 200 μ M, dCTp and dTTP) after, promptly add water to the volume of 100 μ l and the Tag polysaccharase of 2.5units.Reaction conditions is 94 ℃, 1 minute; 37 ℃, 2 minutes; 72 ℃, 3 minutes is a circulation, all carries out 25 segments that circulate and get core protein gene in a large number altogether.The sequence that Sal I is arranged because of 3 ' end design of its C2 introduction, so with PCR increase the generation cAg cDNA segment with Sal I restriction enzyme digestion after, promptly insert the expression vector pG408N that cut with PvuII and Sal I, have λ P promoter on it, and obtain new plastid pG408N-C (see figure 2).Actual gains 2:HCV cAg induction expression of protein

By the pG408N-C plastid of gained in the example 1 at first our method (J.Mol.Biol.165:557-580,1983) of adopting Hanahan with intestinal bacteria earlier under 30 ℃ of conditions the M9-CA substratum with 5ml (contain in 1 liter: Na ₂SO ₄6g, KH ₂PO ₄3g, NaCl 0.5g, MgCl ₂1g, above-mentioned through preparing, accent pH to 7.4 after sterilization, adds following again

Sterile solution; (1M MgSO ₄2ml, 1M CaCl ₂0.1ml, 20% glucose 10ml, 20% Casamino Acid 10ml) overnight incubation, be inoculated in the fresh M9-CA nutrient solution of 10ml with 1/20 ratio again in second day, continuation was cultivated about 3-4 hour at 30 ℃, reach about OD value=0.3-0.5 up to cell concentration, then thalline was placed 42 ℃ of Water Tanks with Temp.-controlled 15 minutes.Thalline after Overheating Treatment continues to cultivate at 37 ℃ again, and 3 hours, can the centrifugation hypothallus.More than through having the proteinic generation of a large amount of cAgs in 3 hours in the thermoinducible intestinal bacteria.Otherwise, do not produce if only in the thalline of 30 ℃ of cultivations, then there is this protein.Utilize the analysis of policapram gel electrophoresis, this result (Fig. 3).This shows that this proteinicly drives expressed come out by the Promoter promoter really.The proteinic analysis of example 3:HCV cAg

Because HCV protein does not still have the production of any unit antibody, so in order to identify whether protein prepared in the example 2 is the relevant antigen protein of HCV, we can only carry out the analysis of west ink marks method with the sufferer serum that has been defined as C type hepatitis through the calibrating of inferior training testing reagent and clinical effectiveness.At first we get 1ml respectively at 30 ℃ of intestinal bacteria liquid of cultivating or luring through 42 ℃ of heat, through centrifugal, and the precipitation hypothallus, again with 2Xcracking, damping fluid (100mM Tris-HCl, pH6.8; 2%SDS; 20% glycerol; 20% beta-mercaptoethanol; 4mM EDTA; 0.01% tetrabromophenol sulfonphthalein) reconstituted, make cell concentration reach OD=10.Boiled then 5 minutes, centrifugal disgorging, take out the 12.5% policapram glue that 15 μ l add the ready 0.1%SDS of containing, use electrophoretic method, the protein that varies in size is separated, immediately the protein on the glue is transferred on the nitrocotton paper (nitrocellulose) according to its relative position then.

This nitre artifical fibre paper is used 5%BSA/TBS (0.05M Tris pH7.6 again; 0.9%NaCl) do and blockade, prevent antibody random adsorption other blank spaces to fibrous paper.Then the nitrocotton paper of handling and patients serum's (with 3%BSA/TBS dilution in 1: 500) are made association reaction.Reacted nitrocotton paper uses scavenging solution (0.25%Tween-20/TBS) to clean (vibration gently, each 10 minutes) 6 times at once, does not have and antigen protein bonded antibody to remove.The nitrocotton paper that cleaned is made association reaction for the second time with the anti-human Ig antibody (with 1: 3000 times of dilution of 3%BSA/TBS) of the goat that is connected to alkaline phosphatase esterase again.After the reaction, clean 6 times with above-mentioned scavenging solution equally, use buffer A (100mM Tris-HCl, pH9.5,100mM NaCl.50mM MgCl at last again ₂) clean once, be subjected to matter solution (10ml buffer A+40 μ lNBT solution+35 μ l X-phosphate solutions) to what nitrocotton paper was dipped in 10ml then, (NBT solution: the diformazan with 70% is for the nitroblue tetrazolium salt that is mixed with 75mg/ml with acid amides (V/V); The X-phosphate solution: the diformazan with 70% is for the 5-bromo-4-chloro-3-that is mixed with 50mg/ml with acid amides (V/V)

The base phosphoric acid solution), do color reaction.After nitrocotton paper colour generation is suitable, can add TE solution (10mM Tris, pH8.0,1mM EDTA) stopped reaction, clean with water more at last, get final product.Example 4:

Via the method for being narrated in the above-mentioned example, utilize pG408N-C to express plastid in this example and the escherichia coli host that contains cI 857 genes prepares the HCV cAg albumen that a kind of C of detection type hepatitis is used.Account for 10% (Fig. 3) of thalline all protein through the prepared cAg protein output of method thus.This protein really can be positive with the patients serum who through clinical diagnosis is C type chronic hepatitis, but not with normal people's sero-reaction (Fig. 4), 63 chronic hepatitis patients serums that we are further provided by the platform large hospital (all detecting the reaction of its anti-C100-3 through inferior training reagent) verify the effect of core protein.Found that wherein has 46 result's (76%) of (35 positives, 11 feminine genders) is consistent with Ya Pei reagent place detected result; And 11 serum samples are arranged in addition under the situation that training reagent in Asia can't sensitive measure, but can very strong association reaction (seeing Table 1) be arranged with our prepared cAg albumen.Because these patients have all presented the chronic hepatitis symptom clinically, so show that only training reagent with the Asia detects, and truly have fish that has escape the net, and the prepared cAg of the present invention can remedy this defective.Certainly also have the inferior training of 2 samples reagent to measure greater than 2, other has 4 samples, detects at Asia training reagent to be low reaction person (within 1.0＞OD＞0.4 scope), and does not partly have significant reaction person at cAg; This may be because the antibody that the result within the limit of error of inferior training reagent or this six patients do not have anti-core as yet produces; But these deductions still remain to confirm it with additive method.Most importantly the result who utilizes this antigen protein and be diagnosed matches (seeing Table 1) substantially and by the diagnosis person of Ya Pei reagent place, and cAg of the present invention still can obviously detect 11 Ya Pei reagent place can't detected serum sample.Other 12 normal peoples' the serologic test result reaction that all is negative.Generally speaking, utilize the reagent of cAg, can remedy the part that number of C 100-3 testing reagent is not detected, a more not false-negative diagnostic result of tool is provided as testing C-type hepatitis.Table 1: it is anti-to analyze NANB hepatitis sufferer antagonism HCV core with west ink marks method

Former antibody response

Whose anti-HCV (C100-3) (a)	Hepatitis (b)	West ink marks method resistive (c)
			O.D＞2.0	Chronic	14/16
2.0＞O.D.＞1.0	Chronic	13/13
			1.0＞O.D.＞0.4	Chronic	8/12
Anti-C100-3 feminine gender	Chronic	11/22
			Normal people's comparative group		0/12

Annotate: a. trains reagent with the Asia, anti-C100-3, the reaction result of gained.

B. the hepatitis situation of clinical diagnosis.

C. the west ink blok response analysis that refers to the cAg of sufferer serum antagonism HCV

The result.

Claims (3)

1. method of utilizing intestinal bacteria preparations to can be used to the antigen protein of testing C-type hepatitis virus HCV is characterized in that described HCV antigen protein has following aminoacid sequence:

Met?Ser?Thr?Asn?Pro?Lys?Pro?Gln?Arg?Lys 10

Thr?Lys?Arg?Asn?Thr?Arg?Arg?Arg?Pro?Gln 20

Asp?Val?Lys?Phe?Pro?Gly?Gly?Gly?Gln?Ile 30

Val?Gly?Gly?Val?Tyr?Leu?Leu?Pro?Arg?Arg 40

Gly?Pro?Arg?leu?Gly?Val?Arg?Ala?Thr?Arg 50

Lys?Thr?Ser?Glu?Arg?Ser?Gln?Pro?Arg?Gly 60

Arg?Arg?Gln?Pro?Ile?Pro?Lys?Ala?Arg?Glu 70

Pro?Glu?Gly?Arg?Ala?Trp?Als?Gln?Pro?Gly 80

Tyr?Pro?Trp?Pro?Leu?Tyr?Gly?Asn?Glu?Gly 90

Leu?Gly?Trp?Ala?Gly?Trp?Leu?Leu?Ser?Pro?100

Arg?Gly?Ser?Arg?Pro?Asn?Trp?Gly?Pro?Thr?110

Asp?Pro?Arg?Arg?Arg?Ser?Arg?Asn?Leu?Gly?120

Ter; Its preparation method comprises the steps:

(a) the cDNA segment of synthetic one section HCV;

(b) from the cAg cDNA segment of the synthetic HCA of the cDNA segment of this HCV;

(c) P1 of this cAg cDNA segment being cloned into the expression plastid pG408N that cut with Pvu II and Sal I transcribes into promoter, and the plastid pG408N-C that obtains;

(d) change resulting pG408N-C plastid over to contain c I857 gene intestinal bacteria;

(e) this bacterium is cultivated down at 30 ℃, bacteria concentration is warming up to 40-43 ℃ again to 0.D680=0.3-0.5;

(f) cultivate down in 36-38 ℃ again;

(g) separating thallus and obtain the antigen protein of HCV.

2. according to the described method of utilizing the intestinal bacteria preparation to can be used to the antigen protein of testing C-type hepatitis HCV virus of claim 1, it is characterized in that step (e) is again that degree of intensification is to 42 ℃.

3. according to the described method of utilizing the intestinal bacteria preparation to can be used to the antigen protein of testing C-type hepatitis HCV virus of claim 1, it is characterized in that step (f) is to cultivate down at 37 ℃.

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