CN1072454A - Recombinant avirulent salmonella antifertility vaccines - Google Patents

Recombinant avirulent salmonella antifertility vaccines Download PDF

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CN1072454A
CN1072454A CN91108013A CN91108013A CN1072454A CN 1072454 A CN1072454 A CN 1072454A CN 91108013 A CN91108013 A CN 91108013A CN 91108013 A CN91108013 A CN 91108013A CN 1072454 A CN1072454 A CN 1072454A
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bacterial strain
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柯蒂斯Ⅲ·罗伊
滕恩·肯尼思S·K
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University of Washington
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Abstract

Invented the non-toxic microbe that comprises coding gamete specificity antigenic recombinant expression system, this microorganism can be used in the vaccine component, and immune a kind of vertebrates makes it anti-gamete specificity antigen, prevents or reduce the person's that accepts the vaccine conceptual quotient thus.

Description

Recombinant avirulent salmonella antifertility vaccines
The governmental fund explanation
The present invention is by government-funded: fund numbering RO1 DE0669, subsidized by NIH (National Institate of Health); Fund numbering CSA-90-071 is provided by contraception research and development plan (CONRAD).United States Government has respective right to this invention.
Technical field
The present invention is main with the antifertility vaccines component and use relevant.Say that specifically this invention means with non-toxic microorganism transmits gamete specificity antigen.
Background of invention
Know to have the normally sterile or fertility reduction of masculinity and femininity that remarkable anti-people's sperm antibody is tired, and do not have other illness (Ingerslev and Ingerslev, 1989 already; Chen and Jones, 1981; Menge et al., 1982; Bronson et al., 1984).Also proved with the total extract immunity of sperm is male and can cause sterile (Kummerfeld and Foote, 1976 with jenny; Munoz and Metz, 1978; Tung et al., 1979; Menge et al., 1979).Primakoff etc. (1988a) come the single-minded surface antigen of a kind of guinea pig sperm of purifying with monoclonal antibody, PHG-20, and prove that the male or female cavy of antigen injection with this purifying all causes secular anti-fertilization immunity (1988b).Former experiments shows that this monoclonal antibody and sperm adhere to plain reaction, hinders it and the interaction of ovum zona pellucida, and this interaction is the important step (Primakoff et al., 1985) of being fertilized.
Have been found that spermatogeny by many other monoclonal antibodies of the sperm preparation of people's sperm or other species and people intersects that send out should.The component reaction of some of them and seminal fluid, the antigen of other identification tool testis origins.Reported and can make people's sperm outage or gathering, perhaps suppress sperm in conjunction with and penetrate the monoclonal antibody of hamster no pellucid zone ovum.Current, the sperm antigen of having known some is as the antigen (Moore et al., 1987) of the molecular weight 95,000 found by Moore; By Lee find by S36-37 mAbs(HSA-63) antigen (Liu et al., 1990) of the 55KDa of identification; The people's of the sperm receptor of the ZP-3 of 95KDa and 56KDa analogue ((Bliel, 1990 of in mouse, identifying by Saling.Bliel and Wassarman; Leyton and Saling, 1989)).Interesting equally is mouse and people's FA-1 antigen; By the 24KD antigen of Naz part qualitative (Naz, 1988) and rat and people's testis, by Shaha qualitative (Shaha et al., 1990).The antigen that Moore and Lee find, and SP-10 immunogen (below will illustrate) is decided to be " primary candidate vaccine " (Anderson et al., 1987) by fertility control vaccine working group of the World Health Organization (the World Health Organization Taskforce on Vaccines for Fertility Regulation).
Sperm specificity antigen-serum lactic dehydrogenase C(LDH-C), be purified, qualitative, and be used at rabbit (Goldberg et al., 1973), mouse (Lerum and Golderg, 1974) and on the baboon (Goldberg et al., 1981) carry out immunity and suppress fertility.This sperm specificity LDH-C has the substrate specificity different with heart LDH with muscle, and it does can also make substrate with branched-chain keto acids the substrate except utilizing lactic acid, as alpha-ketoisocaproic acid.LDH-C is present in (Montamat et al. in the tenuigenin and plastosome of sperm, 1988), but the surface (Erickson et al., 1975) that also is present in sperm, this is providing the foundation for the validity of the anti-LDH-C immunity that stops fertilization just.Recently, Goldberg and its colleague have cloned the cDNA of the narrow spectrum serum lactic dehydrogenase of people's testis, and have determined its antigen decision site (Millan et al., 1987; Hogerfe et al., 1987; Goldberg, 1987; Hogerfe et al., 1989).
A kind of LDH isozyme LDH-X that finds in male spermoblast solves the most clearly human sperm antigen.It has also been known its amino-acid sequence (Goldberg by crystallization, 1972), in mouse and rabbit, found to LDH-X reply (Goldberg, 1972) from body and alloimmunity, though as if LDH-X is not that an effective autoimmunity is former in the people.By systematically or at uterine region inoculation LDH-X making baboon sterile (Samuel et al., 1978).
People such as Wright (1989) have identified the λ gt11 clone of the single-minded acrosome internal protein antigen SP-10 of expressing human sperm.This antigen be present in the sperm of high primates and pig (Herr et al., 1989b).By with monoclonal antibody NHS-10(Homyk et al., 1989) reaction to identify this SP-10 antigen molecular be 28.3KDa.This SP-10 antigen only just is positioned sperm surface after acrosomal reaction, may be at this moment, thereby the SP-10 antigenic action of antibody and exposure suppressed to cause the sperm-zona pellucida of being fertilized interact (Her et al., 1989b).
A kind of SP-10 fusion rotein by 640 nucleotide codings is that the part by the beta galactose thuja acid of the fragment in the immunogen district that comprises the SP-10 molecule and bacterium is formed by connecting, and this fusion rotein has been done the immunogenicity test on rabbit.Rabbit produced can with the polyclonal antibody from the extractive natural SP-10 effect of people's sperm.These antibody people's perforatorium that can dye.Do not show any illness (Benjamin, D.C., et al., 1985) after these rabbit vaccinations.
In addition, the generation of immunoglobulin A, the chances are secretory IgA (sIgA), ability (the Kremer and Jager that sperm has penetrated cervical mucus will be hindered, 1980), and suppress interaction (Dor et al., 1981 of sperm-zona pellucida in the fertilization process; Bronson et al., 1982a, 1982b).So a kind of immunization method that remove to stimulate body fluid and cellullar immunologic response external enwergy to stimulate sIgA to reply will be very good.
Ovum specificity zona pellucida antigen ZP-3, the antigen that this exactly sIgA of causing replys, this antigen reply and cause that sIgA covers zona pellucida, thereby prevent the sperm fertilization.ZP-3 is special to be present in ripe and in the sophisticated ovum, and to sperm in conjunction with and induce acrosomal reaction very important (Wasserman, 1987).All make its ovarian function undesired or forfeiture ovarian follicle (Wood et al., 1981 with porcine zona pellucida or ZP-3 immunize rabbit, dog and monkey; Mahi-Brown et al., 1982).Yet, with ZP-3B cell antigen determinant mouse is carried out non-intestine immunity with the fusion of keyhole limpet hemocyanin, can cause that the Switzerland mouse is sterile with reversible completely, but cause the female mouse ovary of B6AF1 autoimmune disease and complete irreversible sterile (Tung et al., 1991).Recently, may induce the mouse ZP-3 epitope of reversible sterile immunne response to separate (Tung et al., 1991) this with the epitope of inducing the autoimmunization ovaritis.In vaccine, use the effective ways that this peptide species can provide the blocking-up fertilization, and do not produce adverse consequences.
It is to introduce receptor with nontoxic carrier microorganism that yet above-mentioned antigens does not also have a kind of.Shown that some nontoxic carrier microorganisms that contain exogenous antigen can cause excretory, body fluid and immunity cell.In these bacterial strains, import sudden change, be enough to make bacterium can not be created in the required protein that function is arranged of existence among the host.That is to say that these bacterial strains can not in some way or continue for some time in the host survives, thereby can not cause that the host is impaired or pathogenic.These existing reports of sudden change ((EPO Pub.No.315,682(1989/5/17 delivers), PCT Pub.No.Wo 88/09669(1988/12/15 delivers), Curtiss and Kelley, 1987)).Its representative is Salmonellas (Salmonella spp.) mutant strain, its deletion mutantion influences bacterium synthesizing adenosine cyclase of acid (the ATP tetra-sodium splits and closes (cyclisation) enzyme, EC 4.6.1.1.) (cya) and the ability of cyclisation AMP receptor protein (Crp).And, remove mouse and hinder plug Salmonellas typhimurium) in the toxicity plasmid (Jones et al, 1982) of 91R6 can eliminate toxicity and lethality after oral effectively.
Also set up simultaneously the mutant strain that contains coding β-aspartate-semialdehyde dehydrogenase (asd) point mutation or disappearance teat.This kind of enzyme is present in a diaminopimelic acid (DAP) route of synthesis.DAP is an important component of peptidoglycan.DAP can keep the form and the soundness of bacteria cell wall.The bacterium that contains the asd sudden change can only survive under the laboratory environment of strictness control.So, can be the asd(Asd that encodes simultaneously +Carrier) and desired antigenic recombinant vectors introduce Asd -Carrier's cell in.Only containing the required antigenic cell of coding could survive.Transmit the effective ways that sperm specificity antigen will be Birth control with such carrier microorganism.
Content of the present invention
The present invention is based on such discovery, promptly some non-toxic microorganism can be as sperm specificity and the antigenic carrier of ovum specificity.These microorganisms are very useful in anti-fertilization vaccine.These vaccines provide effective means to prevent to accept the immune to become pregnant.
Thereby a project of the present invention is a kind of non-toxic microorganism, and it contains the antigenic recombinant expression system of at least a gamete specificity of coding.
Another project of the present invention is a kind of vaccine component, and it contains the non-toxic microorganism of uniting the effective in cure dosage of use with pharmaceutically acceptable carrier.
In the project that especially will point out, non-toxic microorganism lacks the natural dyeing body gene of coding β-aspartate-semialdehyde dehydrogenase (asd), and contains the recombination that coding has the asd polypeptide of function.What link to each other with this recombination is one or more gamete specificity antigenic genes, particularly LDH-C of coding, SP-10 and/(perhaps) ZP-3, or their epitope.This non-toxic microorganism contains the cya mutator gene simultaneously, and the result can not produce the adenylate cyclase of function in fact, also contains the crp mutator gene, makes them can not produce the cyclisation AMP receptor protein of function.
The invention of this special project also has a project, can be used for inducing the vertebrates acceptor to be in anti-fertilization status.This method comprises that the above-mentioned vaccine with effective dose imposes on acceptor.
These projects of the present invention and other project are all used routine techniques this area from it seems here.
The chart brief description
Figure I shows the cDNA order and the side order (Wright etc. of the coding region of the SP-10 on λ gt11 clone SP-10-5 and the SP-10-10; 1989).In the Hexanucleotide recognition sequence that 5 of SP-10-5 order starting point ' 61 Nucleotide places of end are EcoRI, 3 ' end, the 1091 bit base places of SP-10-10 order are the Hexanucleotide recognition sequence GAATTC of an EcoRI.SP-10-10 has the disappearance that does not change reading frame of one 57 base pair in proper order, is marked by bracket.The restriction enzyme recognition site that is used to make up is understood in the figure acceptance of the bid.The excision site of arrow indicator signal order.
Fig. 2 shows Asd +Cloning vector PYA292.
Fig. 3 is Asd +Cloning vector pYA810.This carrier contains the trc promotor, multiple clone site, the replication origin of rrnB transcription terminator and p15a.Clone the control that is subjected to the trc promotor in its expression of gene of multiple clone site.
Fig. 4 shows nucleotide sequence and the multiple clone site of the Ptrc of pYA810.
Fig. 5 shows Asd +Carrier pYA3042, it contains the gene of the coding people sperm specificity LDH-C of the trc promotor promotion that is subjected to pYA810.Shown among the figure and be inserted in eight amino acid (runic) SmaI-Hind III site among the pYA810, that increase on the LDH-C gene from pHam-LDH-C.
Fig. 6 shows lacI qMaterial grain pYA232, lacI are met in resistance qGene is carried by the psc101 replicon, and therefore in the balanced lethal system host-vector system, the expression of structure gene is subjected to Dtrc promotor (Ptrc) and the control of relevant promotor on the p15A replicon.
Fig. 7 shows plasmid pKKHC4, and it contains the open reading frame of 966 base pairs of coding people sperm specificity LDH-C.
Fig. 8 shows that the 1.1Kb EcoRI-Hind III fragment cloning that comprises the LDH-C gene from pKKHC4 of plasmid pYA3054 is on the EcoRI-Hind of pYA810 III site.
Fig. 9 shows plasmid pYA3048's.9A represents that the LT-B gene clone is between the BanHI and PstI site of pYA810.LT-B gene 3 ' end has the joint of 38bp, contains BanHI, MluI and the ApaLI at single point of contact.9B represents the multiple clone site that LT-B order C-does not hold among the pYA3048.The C-of LT-B not terminal amino acid is Asn.* represent two terminator codons, each is in different reading frames.
Figure 10 shows co-expression plasmid pYA3095 and the pYA3097 of LT-B and LDH-C, and building process.
Figure 11 shows the LT-B-people LDH-C fusion plasmid that is made up by pKKHC4 and pYA3082.
Figure 12 shows the structure of MBP-LDH-C fusant, and the N-that shows the pMAL-cNOT carrier having with three amino acid whose LDH-C genes that are added on after the Xa factor cutting on the LDH-C of petiolarea and pKKHC4 not.
Figure 13 shows from the number of the colony-forming unit (CFU) of the reorganization Salnonella of the expression LDH-C of mouse recovery, by example nine explanations.
Figure 14 shows the structure of pYASP-10-5+.The non-SP-10 amino acids coding order of EcoRI site 3 ' end can remove by the polynucleotide sequence that inserts two aspartic acid codons and a terminator codon that is right after, thereby obtains pYASP-10-5ter.
Figure 15 shows the structure of pYASP-10ter.
Figure 16 shows the structure of pYALT-B-SP-10.
Figure 17 shows the structure of LT-B-ZP-3 fusant, and shows the coding region of mouse ZP3 clone pZP3.3, and toxicity epitope zone and synthetic oligomer.The relative position (base pair) of numeral point of contact under the restriction enzyme site on ZP3.The numeral of magnification region top shows amino acid whose position (amino acid no).
Figure 18 shows pYA3111LT-B-ZP-3 tenuigenin fusion rotein recombinant plasmid, and it is formed by the MluI site that the synthetic oligonucleotide annealing of 50bp is connected into pYA3082.
Figure 19 shows LT-B-ZP3 pericentral siphon fusion rotein recombinant plasmid pYA3112, and it is the ClaI-PstI site formation that the ClaI-PstI fragment of pYA3111 is connected into pYA3048.
Figure 20 shows asd +Expression vector pYA3098 is used to make up pYASP-10Nter and pYASP-10Cter.
Detailed explanation of the present invention
Implementation of the present invention only needs to use the routine techniques of cells involved cultivation, microorganism, restructuring DNA and immunology aspect except as otherwise noted. These technology have detailed explanation in the literature. Such as Sambrook etc., Molecular Cloning:A laboratory Manual, Second Edition(1989) Vols 1-3; DNA Cloning(1985) Vols I and II, D.N.Glover(ed.); Nucleic Acid Hybridization(1984), B.D.hames, et al.(eds.); Perbal, B., A Practical Guide to Molecular Cloning(1984); Metheds in Enzymology(the series), Academic Press, Inc; Vectors:A Survey of Molecular Cloning Vectors and Their Uses(1987), R.L.Ro-driguez, et al., Experiments in Molecular Genetics(1972) Cold Spring Harber Laboratory.
All patents of mentioning above and hereinafter, patent application and publication, hereby row In list of references.
A. definition
" antigen " refers to contain the molecule that one or more antigens determine base, and its energy stimulation of host immune system generation is secreted, antigen selectivity body fluid and cell is replied. This term can with " immunity former " Alternate.
" gamete selectivity antigen " in this article refers to the antigen that the immunity that can cause anti-ovum or sperm is replied. " sperm selectivity antigen " can cause the immunity of anti-sperm and replys, and " ovum selectivity antigen " then causes the immunity of anti-ovum and replys. This kind gamete selectivity antigen might not derive from by the acceptor of immunity, and as long as they can cause required immune response. The example that several gamete selectivity antigens will be arranged below.
" induce anti-fertility state " and refer in acceptor, set up immunity and reply, the fertilization rate of acceptor is reduced or the fertilization effect is prevented from than normal condition. The anti-fertility state of this kind might not be permanent, also can be reversible. Yet the present invention also is concerned about irreversible anti-fertility state (also just referring to sterile).
" haptens " is meant the molecule that contains one or more epitopes, but it self can not activate host immune system generation secretion, body fluid or cellullar immunologic response.
" epitope " is meant can be by specificity antibody molecule bonded position on antigen or hapten molecule.This term also can exchange with " antigenic determinant " or " antigen decision site " and use.An epitope generally comprises necessary three amino acid of identification space structure, five amino acid more commonly, and what occur at most is 8-10 amino acid." epitope " in this definition can cause immunne response in the receptor at it.
" immunne response " to a component or vaccine is meant in the host this component or the cell of vaccine generation or antibody-mediated immune response.Usually, this replying comprise by receptor and produce antibody, and the B cell helps the T cell, and suppressor T cell and cytotoxic T cell are as to comprising antigenic reaction in antigen or vaccine and the component thereof.
" vaccine component " is meant a kind of material, and it can be used to stimulate the immunity system of an organism, this organism is protected and avoids following possible fertilization." immunization " is meant the process that continues high-level antibody and cellullar immunologic response that produces of inducing, in cellullar immunologic response, the T lymphocyte can make the antigen inactivation, perhaps activating other cell (for example phagocytic cell) in body comes inactivation antigen, antigen herein to be meant body contacted antigen in the past.Though " immunity system " comprises the immune response of unicellular organism to external individuality, for example: the generation of Interferon, rabbit, and this phrase only refers to a kind of anatomical structure and the mechanism of multicellular organism in this article, can make this biology produce corresponding antibody when cell or extracellular fluid suffer the antigenic substance invasion.The antibody of Chan Shenging can belong to any immunization type like this, immunoglobulin A for example, D, E, G or M.
Interesting especially is to stimulate the vaccine that produces immunoglobulin A (IgA), because this is the main immunoglobulin that is produced by the warm blooded animal excretory system.Most of pathogenic agent are assembled or invasion at mucodermis.Various glands produce secretory IgA (sIgA), and appear at respiratory tract, in the secretory product of the infiltration mucodermis of intestines and stomach and reproductive tract, thereby stop the assembly and the invasion on the surface of specific antigen.A lot of to antigenic immunne response research, report also a lot, in " the immunology teaching material " of Barrett and James T., immunology there is a general introduction (Textbook of Immunology:Fourth Edition, C.V.Mosby Co., St.Louis, MO(1983)).
A kind of " dosage " of vaccine component is meant is enough to weaken or block fertilization dosage in the vaccine receptor.The dosage of currently available vaccines, existing vaccines component can be by routine the method for control experiment determine.Doses vaccine treatment group is compared with control group, just can know whether this dosage can stop or reduce fertilization effectively.In general, effective dose changes with the method for injecting vaccine is different.Proper dosage will be discussed in the back.
" vertebrates " is meant any animal in the Vertebrata, is the major portion of vertebrate, comprises fish, batrachians, reptiles, birds and mammals, and the common trait of all these animals is to have backbone merogenesis, os osseum or cartilage.All vertebratess all have the immunity system of function, and carry out immunne response by producing antibody.
" individuality " or " receptor " of accepting vaccine among the present invention comprises all vertebratess in this article, and for example, mammals comprises livestock and people, and various birds comprise poultry, particularly those poultry significant on agricultural.
When " the nontoxic derivative strain of microorganism " is meant this microbiological treatment host, can not cause host disease.Non-toxic microorganism in this article, is the derivative strain of pathogenic micro-organism, and can moves and be born in the lymph reticular tissue." cause of disease " is meant and can causes disease or infringement normal physiological function.Avirulent strain can not resemble diseases induced comprehensive symptom its corresponding poisonous pathogenic micro-organism.Term " microorganism " comprises bacterium in this article, protozoon and unicellular fungi.The change strain of non-toxic microorganism also is a concern scope of the present invention " " derivative strain " be meant the sexual or vegetative progeny and the mutant of avirulent strain, comprises that single or multiple base replaces, disappearance is inserted or inversion.
" carrier microorganism " is exactly the non-toxic microorganism that includes and express the recombination of encode specific protein (for example gamete specificity antigen) of top indication.
" recombination " is meant one section certified polynucleotide segment of energy in long polynucleotide molecule, and this segment is not present in the natural molecule of big polynucleotide.
" replicon " is meant any gene that can exist with DNA self-replicating unit in vivo (for example, plasmid, karyomit(e), virus), that is to say that can self control it duplicates.
" carrier " just is meant certain replicon, plasmid for example, and phage or clay duplicate it thereby can connect another dna fragmentation in vivo.
DNA " encoding sequence " is meant the section of DNA sequence, when it is in suitable following time of adjusting sequential control, can be transcribed and translate into polypeptide in vivo.The codon decision is ended by the translation of initiator codon and the 3 ' end (carboxylic acid) of 5 ' end (aminoterminal) in the border of encoding sequence.Encoding sequence comprises the procaryotic DNA sequence, derives from the cDNA of eukaryotic mrna, the genomic dna sequence of eukaryote (for example mammals), even comprise the synthetic DNA sequence.One section transcription pausing order is by being positioned at 3 of encoding sequence ' end.
" initiator sequence " is meant a DNA regulatory region, can transcribe downstream (3 ' direction) encoding sequence in conjunction with RNA polymerase and startup in cell.For needs of the present invention are described, 3 ' termination of initiator sequence has the translation initiation codon of encoding sequence (ATG), and promotor is to 5 ' extension, and the base that comprises minimum number is higher than background level and detectable transcribing with startup.In initiator sequence, can find transcription initiation site (can examine and determine easily) with nuclease S-1 collection of illustrative plates, and in conjunction with the protein binding district of RNA polymerase (conservative order).Eukaryotic promoter usually but always do not comprise " TATA " box and " CAT " box.Prokaryotic promoter also contains the Shine-Dalgarno order except-10 and-35 conservative order.
DNA " control sequence " comprises initiator sequence, the rrna binding sequence, the polyadenylic acid signal, transfection terminating sequence, the upstream regulation district, enhanser or the like, all these make in proper order encoding sequence in host cell transcribe and translation is carried out.
In cell, encoding sequence is conditioned order " manipulation " or " control ", and RNA polymerase is attached on the initiator sequence and with encoding sequence and is transcribed into mRNA, and mRNA is translated into the polypeptide that encoding sequence determines then.
" recombinant host cell ", " host cell ", " cell " and some other refer to that the term of microorganism is exchanged use, mean the microorganism cells that can or be used as the acceptor of recombinant vectors or other transfer DNA, also comprise the offspring of transfected naive cell.Know now, because the offspring of the chance or the parental cell of sudden change of having a mind to might not be consistent with genome and total DNA of parental cell.Daughter cell and parental cell have enough similaritys, and can differentiate its correlation properties, and the natural gene of the key enzyme of for example encoding is replaced by the structure gene of a clone's expression specific protein.
" clone " is by the unicellular or cell mass that the common ancestor produces by cell fission." clone " is meant clone's primary cell, and it can stably be grown many generations external.
" gene library " is meant all perhaps polygenic set of cloning from a specific kind.The acquisition in library is the restriction enzyme processing DNA that selectes by using, and dna segment is cloned on the suitable carriers.Gene library can be screened with the dna homology sequence that obtains from related organisms, thereby isolates the clone of the required gene of representative in the library.
" allos " district of dna structure is meant an identifiable dna segment, and it is arranged in another dna molecular or links to each other with another dna molecular, and does not have related with the native sequences of this DNA.Like this, if bacterial gene of an allos district coding, the dna sequence dna of these gene both sides is not present in the genome of gene source bacterial strain.The example of another allogeneic coding sequence is a kind of nature and non-existent structural coding sequence (synthetic gene that for example, has the codon different with natural gene).And allelotrope changes and spontaneous mutation does not produce DNA allos district, and is pointed just as this paper.
" conversion " mean in this article in host cell and to insert the external source polynucleotide, no matter and what the method for inserting is, for example: directly absorb transduction or joint.Exogenous polynucleotide can exist with the plasmid form, perhaps, is incorporated on the host genome.
B. general method
The present invention is with to contain the antigenic microorganism vaccine of gamete specificity relevant, can cause amphigameously weakening or eliminating on the receptor of vaccine administration.Now known multiple sperm specificity antigen, PHG-20 for example, SP-10, LDH-C, LDH-X, the M of Moore 195000 antigens, Lee's can be by S36-37mAbs(HSA-63) the 55KDa antigen of identification, the people's of the 95KDa of ZP-3 and 56KDa sperm receptor analogue, Shaha is 24KDa antigen qualitatively, and FA-1(Naz 1987,1988).These specificity antigens will be used in the present invention.The nucleotide sequence of SP-10 as shown in Figure 1.In addition, the cDNA of people's testis specificity serum lactic dehydrogenase has also been known its antigen site by the clone.(Millan et al.,1987;Hogrefe et al.,1987;Goldberg,1987;Hogrefe et al.,1989)。Equally, some ovum specificity antigens also know, zona pellucida antigen for example, and ZP-1, ZP-2, ZP-3, and differentiated the epitope among the ZP-3 (Tung et al., 1991).
These or other the antigenic gene of gamete specificity of encoding can be transferred on the non-toxic microorganism, and is conveyed into suitable receptor.On the microorganism carrier, do not need to contain the antigenic encoding sequence of whole gamete specificity, as long as and contain the gene of at least one epitope, so just can on vaccinated acceptor, cause immunne response.In fact, with regard to ZP-3, if expect the anti-fertilization status of reversible, be preferably in and remove the epitope (pathogen antigen decision base as shown in figure 17) that can bring out autoimmune disease on the albumen, then with the albumen after the disappearance or just with causing sterile immunoreactive epitope to come immune receptor.This epitope is identified (Figure 17 and Tung et al., 1991).When needs obtain permanently when sterile, then can in anti-fertilization vaccine, use the ZP-3 that comprises the cause of disease district.
Other gamete specificity antigen also can be used among the present invention, and available routine techniques is identified.These antigenic genes of encoding can be inserted on the carrier microorganism, resemble that this paper back will introduce, and microorganism transformed can be used for weakening in the vaccine or eliminating the fertilization of acceptor.
In particular, gamete specificity antigen can be identified, prepare, and can their gene of clones coding.At first, can prepare cDNA and set up the cDNA library by separating mRNA from testis or ovary, the antibody for preparing with the gamete primary extract can be used for the recon expression screening.Like this, if the albumen of clonal expression can with these antibody responses, just can do further to identify to these albumen.
To some recombinant cDNAs clone, if its expressed proteins can with the antigenic antiserum(antisera) reaction of anti-people's gamete specificity, can be to suitable expressivity plasmid vector and the overexpression proteantigen with its subclone.After recombinant Bacillus coli cells discharges these proteantigens, come purifying antigen with traditional method for purifying proteins.Anti-this proteic antiserum(antisera) can prepare by the former injection of mouse anti, and such efficient is higher, but if will obtain a large amount of antiserum(antisera)s, but then immunize rabbit prepares.In the screening,, make the Western engram analysis with these antiserum(antisera)s again, thereby identify proteantigen in the early stage with sds page electricity arteries and veins separation of human gamete albumen.By this method, can determine whether the cDNA that clones has comprised antigenic entire coded sequence, and whether consistent in the albumen of clonal expression and the gamete.Certainly, proteic glycosylation also is necessary in the correction gamete.This analytical procedure also can show each antigenic different clone's of coding number, and the cD-NA clone of the same antigenic different piece of coding can be sorted out.
An extremely important very crucial analysis is will determine can or can not to react with other tissue of people with the antiserum(antisera) that the antigen that recon produces prepares.This point is extremely important, if because an antigen except gamete, also is present in other tissue, particularly embryonic tissue of people, that will be unacceptable.In other words, particularly important a bit, be used for the gamete specificity antigen that the recombinant avirulent vaccine of human oral immunity is expressed exactly, can not induce antibody and people's tissue have an effect the particularly ovum of after fertilization and the tissue among the embryo.
The mRNA of the distinctive antigen component of the somebody of institute gamete also may not be represented in above-mentioned cDNA library.Therefore, can be with the another kind of method antigenic dna sequence dna of these gamete specificitys that obtains to encode.This just need be to these antigen biochemical method purifying.
The albumen of purifying can be measured its order with existing method, and for example available Edman edman degradation Edman that goes round and begins again is aided with the HPLC amino acid analysis, determines amino-acid sequence.Some sequential determination methods that also have other now.
The amino-acid sequence that aforesaid method is measured can be used for designing the nucleotide oligonucleotide probe, and screens the gene of coding desirable proteins in genomic library.The method for preparing oligonucleotide probe and DNA library all right and wrong is usually advised, can be with reference to books such as " dna clones ".(DNA-Cloning:Vol I, the same; Nucleic Aicd Hybridization, the same; Oligonucleotide Synthesis, the same; Sambrood, et al., the same).
At first, set up the DNA library, prepare oligonucleotide probe then and be used for from the antigenic gene of library screening coding gamete specificity.Oligonucleotide probe can be synthetic with any proper method, and its order is corresponding to the antigenic amino-acid sequence of required gamete specificity.Because the degeneracy of genetic code usually is necessary synthetic multiple oligonucleotide, comprise the nucleotide sequence in a certain zone of all or a considerable amount of proteins encoded.Therefore, when designing probe, can avoid containing the amino acid whose section of height degeneracy to the greatest extent.In some cases, the probe that preparation is quite grown is comparatively desirable, and makes the repetition that higher degree is arranged corresponding to the nucleotide sequence of peptide sequence, particularly when this sequence is peculiar by desirable proteins.In hybrid experiment, it also is good using two probes (or several probe) corresponding to the gene different zones.The synthetic probe of a large amount of kinds of preparation that makes of automatic few nucleic acid becomes quite convenient.The length of the probe that uses is not crucial, but in general, uses the probe of about 14-20 base pair comparatively effective.Sometimes also use the long probe of 25-60 base pair.
Selected oligonucleotide probe will carry out mark with standard method, for example uses radioactive nuleus thuja acid or vitamin H.The probe of mark is applied in the screening step of back, allows isolating SS-DNA hybridization in strand (SS) probe and the library.Suitable hybridization conditions depends on Several Factors, probe length for example, and probe derives from the kind identical with the library or is to evolve to go up sibship kind nearer or far away.Selecting suitable condition also is a skill in this method.Usually can be with reference to top Nucleic Acid Hybridization one book of having mentioned.Basic demand is that hybridization conditions has enough stringencies, make selective cross only for example make have higher homology (as, be higher than 75%) Shi Caineng hybridizes, and avoids non-specificity combination.A clone hybridization in screened library presents the positive, can confirm that this inserts the gene that fragment comprises the desirable proteins of encoding by restricted enzyme cutting analysis and dna sequence analysis.
In addition, this antigenic dna sequence dna of coding also can the directly synthetic preparation without cloning process.Dna sequence dna can design according to the codon of the specific narrow spectrum aminoacid sequence of gamete of coding.In general, if this sequence is used for expression, the just codon that should select expressive host to finish.By standard method synthetic eclipsed oligonucleotide, couple together and form a complete encoding sequence.Example is seen Edge Nature(1981) 292:756; Nambair et al., Science(1984) 223:1299; Jay et al., J Biol Chem(1984) 259:6311.
In case the DMA sequence of coding desirable proteins is produced or separates and obtains, and just it can be cloned in any suitable carriers or the replicon.Having known now has many cloning vectors, and what need do is exactly to select a suitable cloning vector.Can host transformed there be phage (intestinal bacteria Ecoli) in recombinant DNA carrier that is used to clone and institute thereof, pBR322(intestinal bacteria E.coli), pACYC177(intestinal bacteria E.coli), the pKT230(Gram-negative bacteria), the PGV1106(Gram-negative bacteria), pLAFR 1(Gram-negative bacteria), the non-intestinal bacteria Gram-negative bacteria of PME290(), PHV14(intestinal bacteria E.coli and Bacillus subtilus Bacillus), the pBD9(bacillus), PIJ61(streptomycete Streptomyces), PUC6(streptomycete Streptomyces), YIP5 yeast Saccharomyces), YCp19(yeast saccha-romyces) and bovine papilloma virus (mammalian cell).See DNA Cloning:Vol-s. I ﹠ II, the same; Sambrook, et al., the same; Perbal, B., the same.
The narrow spectrum protein coding sequence of our needed gametes places a promotor, ribosome bind site (being used for bacterium expresses), and under the control of the operator gene of non-need (being referred to as at this: " controlling elements ").To comprise behind the carrier transformed acceptor cell of this expression structure this dna sequence dna can transcribe and produce RNA.This encoding sequence may comprise or not comprise signal peptide sequence or leader.Gamete specificity antigen of the present invention can be expressed as tac or trc promotor with the promotor that works in an endogenous promotor or other the known Gram-negative bacterias.
Gamete specificity antigen can be expressed in the carrier microorganism under a control that only allows expression promoter in immune host.Yet produce this protein if desired in a large number, justacrine except these control sequences, also is necessary to increase the adjusting sequence and makes that the expression of antigen sequence is relevant with the growth of host cell so outside microorganism host.Regulate sequence and be known to the expert of this area, related example comprise by to chemistry or physical stimulation react and open or close genetic expression.The regulatory factor of other kinds as enhancer sequence, also can be used in the carrier.
Target protein also can be with a kind of formal representation of fusion rotein, and promptly an allogeneic amino acid sequence is expressed at the C of fusion rotein end or N end.Example is seen U.S. Patent number 4,431,739; 4,425,437.For example, the antigenic dna sequence dna of coding purpose merges with the dna sequence dna of a kind of auxiliary polypeptide of coding, and this auxiliary polypeptide includes suitable antigenic determinant to strengthen the immune response of purpose antigen secretor type.The example of the auxiliary polypeptide of this class comprises B subunit (LT-B) and choleratoxin B subunit (CT-B) (Elson, 1988 of enterotoxigenic intestinal bacteria synthetic heat-labile toxin; Holmgren et al., 1988).Designed the carrier of these polypeptide of constitutive expression, this carrier also has C-terminal or a N beginning that multiple clone site makes coding purpose antigenic nucleotides sequence be listed in auxiliary sequencel and merges (seeing Fig. 9 A, 9B and 11) simultaneously.
An expression vector is fabricated and makes specific encoding sequence and adjusting sequence be positioned at suitable position, and relative position that encoding sequence is suitable with regulating sequence and direction finally make encoding sequence transcribe under the control of regulating sequence.(just with control sequence bonded rna polymerase transcribe encoding sequence) for reaching this purpose, needs the antigenic dna sequence dna of coding purpose is modified.For example, to modify these sequences in order to keep reading frame sometimes, make it to be connected in the regulating and controlling sequence with suitable direction.Control sequence and other adjusting order can be connected with encoding sequence before inserting above-mentioned cloning vector.Perhaps, on the expression vector that comprises a control sequence and a suitable restriction site, directly the clone advances encoding sequence.
Sometimes be necessary to add the preceding paragraph leader and make the synthetic polypeptide go out from the host internal secretion, these secretion signals are cut subsequently.Leader can be by host bacterium through the excision of translation post-treatment, and example is seen U.S. Patent number 4,431,739; 4,425,437; 4,338,397.Sometimes need antigenic mutant of purpose or analogue.Mutant or analogue can be by in the sequence of coding for antigens excalation, the replacement of the insertion of a sequence or or several Nucleotide and obtaining.For example, the protein that is used for immune host may comprise the epitope that stimulates synergid and stimulate the epitope that suppresses cell.Therefore, the coding latter's nucleotide sequence is necessary to modify or lack.The technology of modified nucleotide such as site-directed mutagenesis are widely understood for this area staff.Example is seen Sambrook.et al., and is the same, DNA Cloning, and Vols. I and II, the same; Nucleic Acid Hybridization is the same.
Many known expression vectors are arranged in prokaryotic organism, and example is seen U.S. Patent number 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; UK Patent Application GB2,121,054; GB2,008,123; GB2,007,675; European patent application 103,395.
According to the host of expression system and selection,, under suitable protein expression condition, can produce gamete specificity antigen of the present invention with above-mentioned expression vector host transformed grown cell.For detecting the immunne response of immunized animal, this protein can separation and purification from host cell.If the expression system secretory protein is in growth medium, this protein is purifying from substratum directly.If protein transduction is transported to periplasmic space, can adopt a kind of technology commonly used " cold osmotic shock ", make it to be discharged in the substratum.If protein is not secreted or is transported to periplasmic space, will from cell lysate, separate.Proper growth condition and recovery method are known to the those skilled in the art.
Protein of this invention and fragment thereof can be used for producing mono-clonal or polyclonal antibody.Polyclonal antibody if desired, with antigen of the present invention, its fragment or the antigen immune of a sudden change, behind bird or the Mammals (as chicken, turkey, mouse, rabbit, goat, horse etc.), handle with known method from the serum of being collected on one's body by the animal of immunity.If contain the serum of target protein matter polyclonal antibody, also comprise other antigenic antibody, polyclonal antibody is used the immunoaffinity chromatography purifying routinely.
Protein of this invention and segmental monoclonal antibody thereof, the person skilled in the art is easy to prepare.General method by heterozygosis knurl manufacture order clonal antibody is well-known, the antibody produced cell system of unlimited breeding can obtain by cytogamy, also can pass through other technologies, as transforming bone-marrow-derived lymphocyte with the proto-oncogene dna direct, or infecting bone-marrow-derived lymphocyte with Epstein-Barr virus, example is seen Schreier, M., et al., Hybridoma Techniques(1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas(1981); Kennett et al., Monoclonal Antibodies(1980); U.S. Patent number 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890 cell strains that produce purpose antigen and fragment monoclonal antibody thereof can screen according to different qualities, promptly by epitope or epitope affinity etc.Monoclonal antibody is separated in the corresponding antigen process very effective in the immune affine technology of utilization.
Produce gamete specificity antigen of the present invention as stated above, can make by immune object to be in anti-state of educating.The nontoxic microorganism of carrying is used to provide this antigen.This method is most suitable, because suitable carry the generation that microorganism can stimulate sIgA.SIgA reaches in various glands at respiratory tract, gi tract, and the generation on the reproductive tract epidermis mucous membrane can prevent some to infect the antigenic assembly and the intrusion of mucodermis.The generation of anti-gamete sIgA is restrained sperm and is penetrated the ability (Kramer and Jager, 1980) of cervical mucous membrane and suppress interaction (Dor et al., (1981 between sperm and zona pellucida in the fertilization process in the reproductive tract; Broson et al., 1982ab).
The recombinant plasmid that contains one or several gamete specificity antigen genes can be introduced among a kind of in several nontoxic bacterial isolateses, and these several avirulent strains all have transgenation, and these genes are essential in the medium-term and long-term survival of host animal by bacterium.Non-toxic microorganism commonly used includes, but are not limited to this, the mutant strain of Salmonellas and intestinal bacteria-Salmonellas hybrid.Bacterial classification commonly used in the salmonella has S.typhimurium, S.typhi, S.paraty-phi, S.gallinarum, S.pullorum, S.enteritidis, S.choleraesuis, S.typhimurium that S.arizona. or S.dublin are nontoxic and S.enteritidis bacterial strain have application widely many hosts.Nontoxic S.gallinarum S.pullorum and S.arizona bacterial strain are particularly suitable for immune birds.S.typhimurium, S.typhi and S.paratyphi are applicable to the mankind.S.choleraesuis is applicable to immune swine, and S.dublin then is used for ox.The structure of these mutant strains is seen patent application series number 0,251,304 and Curtiss and Kelly, 1987.
Using wide especially is above-mentioned cya, crp and asd mutant strain, and these bacterial strains can not produce function corresponding protein substantially in the host, because of growth is checked.Yet other non-toxic microorganism can be used for this invention too.These non-toxic microorganism comprise aroA, aroC, aroD, galE, phoP, cdt, ompR and htrA mutant strain.If use Asd -Mutant strain, the gamete specificity purpose antigen carrier by not only encoded the gamete specificity antigen but also the asd that encodes is transformed into and carries in the microorganism.Thereby, have only those to comprise that required gamete specificity is antigenic carries microorganism and can survive, these microorganisms can be selected in order to using.Fig. 2 is pYA292 Asd +Collection of illustrative plates, this is a carrier, above coding gamete specificity antigenic gene can be cloned in.This carrier is transformed into a kind of Asd subsequently -Carry in the microorganism.The expression of the antigenic recombination of coding purpose depends on a control sequence that links to each other with the asd gene.This chain formation relies on the orientation of two genes on carrier, and two genes are placed under identical controlling elements (identical promotor and the operator gene) control.
The Cya mutant strain and (or) the Crp mutant strain can further suddenly change, preferably complete stool sudden change, a pathogenic gene of control Salmonellas of its next-door neighbour Crp gene.This gene, the sudden change of (cdt gene) reduces the ability that bacterium assembles in deep tissues such as spleen.Has crp when one +The plasmid of gene be arranged in △ is arranged (crp-cdt) sudden change bacterial strain the time, it keeps its nontoxicity and immunogenicity, and is identical with crp mutant strain phenotype with cya.On plasmid, comprise crp +The △ of gene (crp-cdt) mutant strain keep its ability at enteron aisle and GALT formation bacterium colony, but the collection in deep tissues reduces to ability.In example 11, isolating △ (crp-cdt) mutant strain from X3622 at first, its argD and cysG gene all lack, need to add arginine and halfcystine to keep growth; This mutation allele is named as △ (crp-cysG)-10.The mutant that another one comprises less disappearance is separated, and its sudden change does not influence arginic synthetic, and it is present among the X3931, is named as △ (crp-cysG)-14.
Utilize transposon, will suddenly change and from other mutant strains, transfer to the purpose bacterial strain, thereby said mutation can be transferred in the specific microorganism.Transposon can be integrated up in many sites in bacterial chromosome.The summary of the characteristic that transposon inserts and lacks is seen Kleckner et al.(1977) .J.Mol.Biol.116:125.For example, have the tetracyclin resistance transposon Tn10 of (with 5-butyl-pyridinium dioctyl phthalate susceptibility), in many bacteria cultures, comprise intestinal bacteria and S.typhimurium, be used to introduce △ cya and △ crp sudden change, the method of the formation of mutant and detection is seen EPO Pub.No.315 in S.typhimuri-um, 682, another kind of method also is provided in the example below.Utilize Tn10, these sudden changes can be inserted in the different salmonella strains, and particularly those have the bacterial strain of very strong pathogenecity.
The formation of mutant bacteria strain also can be adopted the other technologies of knowing.These technology comprise conventional induced-mutation technique and (or) recombinant DNA technology.Needed mutant is selected according to phenotypic characteristic, and some phenotypic characters will be mentioned in the example below.The method that structure has the carrier of these characteristics has more detailed introduction in following patent: number of patent application No 251,304 awaits the reply; Patent application series number 07/612,001, November 9 nineteen ninety.
For the immunne response that stimulates certain to need, preferably microorganism or gene product are directly imported digestive tube and segmental bronchus, as by oral intranasal administration method, gastric intubation method, or in the aerosol mode, and air bag inoculation (being limited to birds) and the interior inoculation method of tracheae.Other appropriate method comprises that passing conjunctiva inoculates accessory lacrimal glands, inoculation in the breast.Other inject the method for vaccine, as intravenous injection, and intramuscular injection, subcutaneous injections etc. also may be utilized, and being mainly used in stimulates secondary immunne response, will further introduce below.
In general, when expressing that the gamete specificity is antigenic carries microbial process in people or other Mammalss, they can received media type appearance with a kind of medicinal going up.For example, carry that microorganism can be wrapped up by casing or by a kind of suitable class gelatin mass parcel.This method is (Cryz and Gl ü ck, 1990, in G.Woodrow and Mr.Levine known to the those skilled in the art, Nen Generation Vaccines, Marcel Dekker, New York, PP, 921-932).
Carry after microorganism enters in the animal body, antigen need act on the immunity system of animal.This will discharge antigen molecule after carrying microbial death.Naturally also can adopt " seepage " non-toxic mutant, can discharge the pericentral siphon composition without bacteriolyze.Perhaps, the gene of selecting a control antigen to produce makes antigen to be discharged in born of the same parents' external environment before carrying microbial death.
Also available aerosol or in tracheae, inject antigen.Usually comprise a kind of mediator if in tracheae, inject antigen, neither stimulate gas visitor mucous membrane, also significantly do not disturb fibre function for the people.Thinner adopts water, salt solution or other known substances in the present invention.Also comprise sanitas such as butylene-chlorohydrin and benzalkonium chloride with agent in the tracheae, be not limited to this certainly.Add surfactant, can strengthen the absorption of tunica mucosa tracheae target protein matter.
In mouth, in the tracheae, in the enteron aisle or with after the aerosol immunity, can carry out the antigenic injection of gamete specificity again.Express the immune immunity of the initial secretor type of microorganism of gamete specificity gene product, this parenteral immunity can strengthen the immunoreactive expression of secretor type.This enhanced reaction is considered to a kind of secondary, and reaction reinforcement or memory causes the secular immunoprotection to the host.Booster immunization can repeat repeatedly, and effect is remarkable.
When vaccine production became injection to be used for booster immunization, they both can be also suspension of the aqueous solution; Also can be made into the solid form that can in liquid media, dissolve or suspend.Prepared product can be emulsified or immune active ingredient wrap up with liposome vectors.Immune active ingredient is normal to be mixed with the mediator that comprises setting agent.This setting agent can be accepted on medicinal, simultaneously also can be compatible with effective constituent.Suitable medium comprise water, salt solution, glucose, glycerine, ethanol etc. and the mixture between them.In addition, if desired, also can increase a spot of auxiliary substance such as wetting agent, emulsifying agent in the carrier, pH agent or adjuvant are to strengthen the effect of vaccine.The adjuvant of mentioning herein comprises Muramyl dipeptide, avridine, hydrogen aluminum hydride, oils, saponin(e and other materials.The concrete grammar for preparing these preparations, known to the people who knows this area, example is seen, Remington ' s Pharmaceutical Sciences, Mark Publishing Com-pany, Easton, PA, 15th et., 1975.All all comprise the protein of q.s, make processed individuality reach required immunological status with preparation.
The antigen amount that applies depends on the object of processing, the ability of object-immunity system synthetic antibody and the degree of protection of needs.Can make dose response curve with routine test, thereby determine effective dose.When making antigen-immunized animal with specific antigen or antigen fragment or antigen analogues, should use a dosage at least, typically carrying the microorganism using dosage is 1 * 10 6-1 * 10 10Individual recombinant avirulent bacterium/immune object.In order to keep, can make antigen increase or multiple doses to immune object to the antigenic immunological status of gamete specificity.
Sometimes need the while or in turn apply several gamete specificity antigen.This both available one comprise coding a plurality of gamete specificitys antigenic gene nontoxic is carried bacterium, and the also available different bacterium of carrying is finished.
Above-mentioned introduction has illustrated the present invention roughly, and following specific example will help further understanding.These examples are only used for explanation, rather than limit to range of application of the present invention.
The bacterial strain of using among the present invention is deposited
Following bacterial strain is stored in American Type Culture Collection, 12301 Park-lawn Drive, Rockville, Maryland.The numeral that increases later is to add after the viability experiment of carrying out success.Needed expense is paid off.During present patent application, have the right to ratify the personnel that someone can obtain above-mentioned bacterial classification according to 37 CFR 1.14 and 35USC122 patent committee member.After the patent of this application is admitted, provide all restrictions of above-mentioned bacterial classification promptly can cancel to the public.Above-mentioned bacterial strains was preserved 30 years from depositing, or preserved 5 years the request back the last time, or according to the enforceable time limit of United States Patent (USP), preserved more longerly.If unexpected destruction can not be survived or be subjected to a certain bacterial strain, perhaps contain the bacterial strain forfeiture plasmid of plasmid, all will replace with the survival strains of same taxonomy proterties.The preservation thing of herein mentioning is for the ease of understanding, rather than for described specially.In addition, these materials are also mentioned in the reference of this patent.
Bacterial strain date saved ATCC No.
X4072 Oct.6,1987 67538
pYA292Asd +
in X6097 Sep.26,1988 67813
pYA3042
in X3987 Nov.16,1990 68479
3958 Nov.2,1990 55110
X4323 Nov.2,1990 55115
X3926 Nov.2,1990 55112
X3927 Nov.2,1990 55117
X4297 Nov.2,1990 55111
X4346 Nov.2,1990 55113
X3940 Nov.2,1990 55119
X4073 Nov.2,1990 55118
ISP2822 Nov.2,1990 55114
ISP1820 Nov.2,1990 55116
PYA3054
in X3987
PYA3111
in X3987
pYA3112+
pYA232
inX3987
Example one
This example is described by introducing influences the deletion mutantion that cAMP is synthetic and utilize, and the separation of the non-toxic microorganism that obtains, and stable to the mutant strain phenotype, the evaluation of totally nontoxic and high immunogenicity.
Intestinal bacteria that bacteria culture is used and Salmonella typhimurium bacterial strain are listed on table 1A and the B.They are suspended in 1% the small protein peptone that contains 5% glycerine, and quick freezing in dry ice-ethanol, double are kept at-70 ℃.Or be suspended in be kept in the 1% bacterium-peptone that contains 50% glycerine-20 ℃ standby.
The conventional complex medium that uses of substratum has L substratum (Lennox, Vixlogy 1:190-206, (1965) and LB substratum (Luria and Burrous, J.Bacteriol.74:461-476(1957)), ultimate density is that 1.2% Difco agar and ultimate density are that 0.65% Difco agar adds and forms lower floor's agar and upper strata soft agar in the LB substratum.Penassay agar is used for conventional bacterial count.Measure fermentative action rely on add Maconkey lower floor's agar or Eosin methylenum coeruleum agar (Curtiss, Genetics 58:9-54(1968)) and ultimate density be 1% carbohydrate.
Synthetic medium is pressed Curtiss by basic liquid (ML) and basic agar (MA), J.Bac-teriol.89:28-40,1965 add the nutritive ingredient of suitable concn, and the buffer salt solution of gelatin (BSG) (Curtiss, 1965 is the same) is used as conventional thinner.
Transducing phage P22HTint is used for transduction (for Genet, Engi, Adv, Bacterial Genetics CSH Laboratory, CSH, NY 1979 for Davis etc., Aman) LB according to ordinary method.The F+strain of incubated overnight is dilution in 1: 20 in the LB of preheating, and 37 ℃ are rotated cultivation 1 hour, are that 0.01 usefulness P22HTint infects with infection multiplicity.Infected the mixture overnight shaking culture about 15 hours, and added chloroform, 37 ℃ were continued shaking culture 10 minutes, and centrifugal suspension (Sorvall Rc5c, ss-34 rotor, 7000 rev/mins, 10 minutes) removes bacterial debris, comprises the supernatant liquor (Ca.10 of phage 10Ml) add 4 ℃ of preservations of chloroform.12.5 the tsiklomitsin of μ g/ml is used to select Tn10 to insert transduction and the sudden change of Tn-10 inductive.
Select Tn10 disappearance substratum and method to see Malcy and Nunn with 5-butyl-pyridinium dioctyl phthalate, (J.Bacteriol.145:1110-1112(1981)).Tn10 inductive mutant strain, under the tsiklomitsin concentration of 12.5 μ g/ml, 37 ℃ of Lbroth incubated overnight are to about 5 * 10 8(FV/ml) culture subsequently in the preheating Lbroth of no tsiklomitsin substratum 1: 40 the dilution 37 ℃ of aerated culture to titres be 2 * 10 9The cell of CFU/ml proper number is (as 10 7-10 8) in BSG, dilute, spread plate on the substratum that contains 5-butyl-pyridinium dioctyl phthalate was cultivated 5-butyl-pyridinium dioctyl phthalate resistance bacterium colony purifying on above-mentioned selection flat board 48 hours for 37 ℃.Select single bacterium colony, at the tsiklomitsin that contains 12.5 μ g/ml with do not contain on the Penassay agar of tsiklomitsin and grow, and check its sensitive tetracycline.
(Sasco, Omaha NB) are used to infect and immunization experiment the female BALB/C mice of mouse (6-8 week).Animal was fed in an isolated room in previous week of experiment, in the cage of the online net bottom of mouse of experiment usefulness, spread Nalgene filter paper in the cage, subsisted and water, and Animal House keeps 22-23 ℃, illumination in 12 hours.
Zoogenetic infection S.typhimurium strain pathogenic strength is determined by oral (P.O) or intraperitoneal inoculation.The bacterium that is seeded in the mouse is the same with conventional cultivation, overnight incubation among 37 ℃ of broth.Culture is dilution in 1: 50 in pre-temperature broth, and 37 ℃ of aerated culture 4 hours are to OD 600Reach 0.8-1.0.Cell is centrifugal in the GSA rotary head, the Sorvall whizzer, and 4 ℃, 7,000 rev/mins, 10 minutes, be suspended in subsequently on the BSG, make 50 times of cell concentration.Suitable diluent scribbles the dull and stereotyped titre of determining on Penassay agar, containing evaluation Cya/crp phenotype on the Mac Conkey agar of 1% maltose, for all S.ty-phimurium oral vaccinations, mouse stopped to supply water for food in inoculation in preceding 4 hours, gave the sodium bicarbonate of mouse feeding 30ml10% with the microsyringe of 20 μ l in 10-15 minute before oral, 20 μ l sampler feedings, 20 μ l's is suspended in S.typhimurium among the BSG subsequently.The supply that oral back 30 parts of clocks recover food and water.Sickness rate of mouse and mortality ratio are observed more than 30 days.The intraperitoneal inoculation of BALB/C mice, an available 26-gauge3/8 " needle injection 100 μ l are suspended in the S.typhimurium dilution of bacteria among the BSG.Sickness rate of mouse and mortality ratio are observed more than 30 days.
The test of protective immunity is infected the mouse that survived through 30 days with any S.typhimurium mutant strain, at the 31st day oral 10 in initial experiment 3-10 4The morbific S.typhimurium parent strain of the mouse of the wild-type of sesquialter lethal dose.Afterwards, the mouse of grouping was used the pathogenic mutation body immunity of various dose respectively, then at initial immunity back different time, with the pathogenic wild type strain inoculation check of various dose.
Separating of the S.typhimurium bacterial strain of △ cya-12 and △ crp-11 sudden change
S.typhimurium SL1344 bacterial strain similar Curtiss of χ 3339 usefulness and Kelly that wild-type has virulence to mouse, 1987 classical genetic method is carried out genetic modification.Method is from PP1037 transduction primary crp-773 ∷ Tn10 sudden change, reach from PP1002 transduction primary cya ∷ Tn10 and suddenly change to highly pathogenicity and infectious S.typhimurium SL1344 bacterial strain χ 3339, select many 5-butyl-pyridinium dioctyl phthalate resistances then, the deletion mutant of sensitive tetracycline, these mutant strains require totally nontoxic, in mouse, have the strongest immunogenicity, also answer genotype highly stable simultaneously.
The insertion of Tn10 transduction can be simplified in crp and the cya gene, main phage P22HTint lysate, and the lysate that in the S.typhimurium bacterial strain PP1002 of band cya ∷ Tn10 sudden change, prepares by the high titre of preparation in the S.typhimurium bacterial strain PP1037 that has crp-773 ∷ Tn10 sudden change.The lysate that finally obtains with infection multiplicity 0.3 recipient bacterium S.typhimurium χ 3339, is transduceed into tetracyclin resistance with it subsequently, selects by maltose defective type phenotype.Phage-infectation of bacteria mixture was cultivated 20 minutes at 37 ℃, was coated on then that (MI), this agar is added with 1% maltose, the tsiklomitsin of 12.5ug/ml for Difco Laboratories, Detroit on the Mac Conkey agar.Cultivate after about 26 hours for 37 ℃, derive from P22HTint(PP1037) → tetracyclin resistance that χ 3339 infects, the bacterium colony of maltose defective type, derive from P22HTint(PP1002) → tetracyclin resistance that χ 3339 infects, the bacterium colony of maltose defective type, picking is rule on same selection substratum in 0.5mlBSG, obtains χ 3339 derivative strains called after χ 3604(cya ∷ Tn10 respectively) and χ 3605(cry-733 ∷ Tn10) (table 1A)
Bacterial strain χ 3604 and χ 3605 grow in being added with L both substratum of 12.5ug/ml tsiklomitsin, every kind of bacterium 100 μ l product dilution in 1: 10 in gelatin buffer salt solution (BSG), be coated with 10 then and contain 5-butyl-pyridinium dioctyl phthalate (FA) culture medium flat plate (Maloy and Nunn, 1981).Flat board was cultivated about 36 hours at 37 ℃, selected five 5-butyl-pyridinium dioctyl phthalate from every flat board, chose among the 0.5mlBSG, then purifying on the FA substratum.The 5-butyl-pyridinium dioctyl phthalate resistance clone picking that purifying is crossed is in L broth, and 37 ℃ are cultured to muddiness, checks the disappearance (sensitive tetracycline) of Tn10.In 10 plates, in the FA substratum, choose a sensitive tetracycline clone, identify that then complete LPS(is by P22HTint susceptibility for every), auxotrophy or prototroph, the stability of genetically deficient, and the answer of tetracyclin resistance.By this process, from χ 3604, chosen 10 independently △ cya mutant, from χ 3605, isolate 10 independently △ cry mutant.
The genetic stability of non-toxic mutant is used for oral bacterial strain as the vaccine of living, must complete stability aspect its nontoxicity and immunogenicity.When the various extent of dilution (10 of culture that concentrate 50 times 9, 10 7, 10 5, 10 3CFU/plate) 10 △ crp mutant and 10 each bacterial strain in the △ cya mutant independently independently, be coated on and include 0.5% maltose, close disaccharides, wood sugar, (be added with on 22ug halfcystine/ml and the 22 μ g arginine/ml), these substratum can not be kept its growth to the minimum medium of glycerine or rhamnosyl, also do not have the growth of revertant and mutant on flat board.One group is duplicated dull and stereotyped through uv irradiating (5 joules/me-ter 2/ sec), 37 ℃ of dark culturing.Another group is dull and stereotyped 37 ℃ of illumination cultivation.Still there are not revertant and mutant after 48 hours.Done an experiment in addition, checked whether the reverse mutation rate of mutation rate or tsiklomitsin equals the wild parental strain of sensitive tetracycline in the mutant strain of fusaric acid resistance △ cya and △ cry.Under all these situations, all do not observe tetracyclin resistance reverse mutation or muton.
Pathogenic and the immunogenicity of △ cya and △ crp mutant strain
Separate 10 △ crp and 10 △ cya mutons of obtaining and be seeded in the BALB/c mouse body, do not screen and determine minimum toxicity, by the thick La of fur appearance or smooth by oral), appetite, vigor (high or low) reflects symptom.Each independently cya or crp deletion mutantion bacterial strain with-10 9CFU gives five mouse oral.By above-mentioned standard recording mouse.Survivor after 30 days is with 10 8Wild-type toxicity parent strain χ 3339 inoculations of CFU.Have three groups in 20 groups that infect with cya or crp deletion mutantion, five mouse survive after △ cry-11 and the △ crp-10 mutant infection, and can resist 10 fully using △ cya-12 at first 4The infection of the wild type strain of sesquialter lethal dose.Wherein one group particularly, △ crp-10 mutant, in nontoxicity, immunogenicity and stable aspect are particularly splendid.Repeat these experiments, the oral or belly inoculation of the △ cry-10 mutant of any dosage, mouse does not show infected (seeing example 3 tables 6)
The proterties of the mutant strain that filters out
Have △ cya-12 respectively, the χ 3615 of △ crp-10 and △ crp-11 sudden change, χ 3622 and χ 3623 are considered to have minimum toxicity, very high immunogenicity and highly stable phenotype and genotype.The data of the phenotypic character of relevant these bacterial strains see Table 2.Table 3 explanation be these bacterial strains with toxicity parent χ 3339 in nontoxicity, the comparative data of immunogenicity aspect, the data of χ 3604 and χ 3605 and cya ∷ Tn10 and the comparison of crp-773 ∷ Tn10 mutant strain.Except the sudden change of the hisG among the parent bacterium χ 3339 needs to add Histidine, △ crp-10 sudden change makes χ 3622 need to increase arginine and halfcystine.These observed data and the character analysis that further △ crp-10 is suddenlyd change are seen example 3.
Example two
This example describes that structure and these mutant strain phenotypes have the avirulent strains that influence the synthetic and deletion mutantion that utilizes of cAMP are stablized, the characteristics of totally nontoxic and high immunogenicity.
Bacterial isolates intestinal bacteria (Escherichia coli) and mouse Salmonellas (Salmonella typhimurium) bacterial strain are listed in table 1.A and B.The preservation of these bacterial strains is seen described in the example one.
The conventional cultivation of substratum bacterium used complex medium.Bacterial count and discriminating are seen described in the example one.
Transduction and screen with 5-butyl-pyridine-2-formic acid that Tn10 loses substratum and method is seen described in the example one.
The mensuration S.typhirnurium bacterial strain toxicity and the immunogenic mensuration of animal infection and protective immunity are seen described in the example one.
The structure that has the S.typhimurium bacterial strain of △ cyg-12 and △ crp-11 deletion mutantion
Vaccine strains with best efficiency may come from showing the attenuation of remarkable clustering capability and infectious high toxicity bacterial strain.Screening has highly pathogenic S.typhimurium wild type strain such as a SL1344(χ 3339), UK-1(χ 3761) and 798 standard the low LD of mouse toxicity in testing arranged 50Value (seeing Table 4), to the having of the susceptibility of antibiotic, toxicity plasmid, genetic manipulation easily (to phage P22HTint and P 1But susceptibility convertibility and acceptant inductive plasmid) with to the susceptibility of Colicine.
As described below, to the toxic strain SL1344(χ 3339 of wild-type), 798 and UK-1(χ 3761) use with Curtiss and Kelly(1987) described classical genetic method similar methods carried out genetic modification.Method is that transposon Tn10(is had tetracyclin resistance) insert near the △ cya-12 or △ crp-11 mutator gene of S.typhimurium bacterial strain SL1344, by P22HTint chain proterties is transduceed, enter highly toxic S.typhimurium bacterial strain UK-1(χ 3761), 798 and SL1344(χ 3339), select the acceptor have tetracyclin resistance and can not utilize the maltose phenotype then, so just will see described in the example one at mutator gene △ cya-12 among the S.typhimurium bacterial strain SL1344 and △ crp-11() changed high toxicity bacterial strain UK-1 over to, 798 and SL1344 in.The insertion sudden change of using be with the chain Zhc-1431 ∷ Tn10 of △ crp-11 and and the chain zid-62 ∷ Tn10 of △ cya-12, two are inserted sudden change and are not influenced the pathogenic of S.typhimurium separately.
In order to help the transduction with the chain missing gene of transposon, at first prepared the lysate of the S.typhimurium bacterial strain χ 3711 of phage P22HTint lysate that the height of the S.typhimurium bacterial strain χ 3773 that has △ crp-11 and zhc-1431 ∷ Tn10 sudden change tires and another one band △ cya-12 and zid-62 ∷ Tn10 sudden change.The P22HTint lysate that obtains is used for the inherited character transduction is entered wild-type receptor bacterial strain χ 3339,798 and χ 3761.
Be used in S.typhimurium χ 3773(△ crp-11 zhc-1431 ∷ Tn10) in the P22HTint virose bacterial strain of transduceing of breeding, filter out then and have tetracyclin resistance and Mal -The acceptor of phenotype.The infection mixture of phage and bacterium, is got the 100ul sample and is coated on the MacConkey agar plate (Difco laboratory, Detroit, Michigan State) that contains 1% maltose (ultimate density) and 12.5ug/ml tsiklomitsin after 20 minutes 37 ℃ of insulations.37 ℃ approximately insulation is after 26 hours, and choosing has tetracyclin resistance and Mal -The transduttant of phenotype and on same substratum purifying.Derive from 798 transduttant called after χ 3825, derive from the transduttant called after χ 3828 of UK-1.Bacterial strain χ 3773, χ 3825 and χ 3828 genotype are △ crp-11 zhc-1431 ∷ Tn10(table 1.B).After these bacterium are cultivated in containing the LB substratum of 12.5ug/ml tsiklomitsin, respectively with 1: 10 dilution proportion to in the gelatin buffer salt solution (BSG), each sample 100ul is coated on substratum (Maloy and the Nunn that contains 5-butyl-pyridine-2-formic acid, 1981) on, flat board is incubated 36 hours approximately at 37 ℃.Choose and have 5-butyl-pyridine in every kind of bacterial strain-being cloned among the 0.5mlBSG of 2-formic acid resistance, then purifying on the FA substratum.5-butyl-pyridine behind the purifying-2-formic acid resistance clone is inoculated in the LB substratum, is cultured to turbid shape at 37 ℃, checks the existence and the auxotroph proterties of the losing of its Tn10 (sensitive tetracycline), complete lipopolysaccharides (LPS).New bacterial strain called after χ 3876(798), χ 3954(UK-1) see described in the example one with separating of χ 3623(SL1344 △ crp-11) (showing 1.B), preceding two bacterial strains all have genotype △ crp-11 △ (zhc-1431 ∷ Tn10).
Because Cya -And Crp -Identical (the Mal of the phenotype of mutant -, Stl -, Mtl -Deng), have clone's cry +The plasmid pSD110 of gene and ampicillin resistance gene is used to the △ cry sudden change on the of short duration complementary karyomit(e), to identify the △ cyg sudden change of introducing by transduction.Having plasmid pSD110(table 1.B) bacterial strain S.typhimurium χ 3670 in the P22HTint of breeding bacterial strain χ 3623, the χ 3876 and the χ 3954 that are used to transduce and in the LB substratum, cultivate.Selecting substratum is Mac Conkey agar+1% maltose+100ug penbritin/ml.After 26 hours, each bacterial strain is chosen a tool amicillin resistance and Mal +Purifying on the flat board that is cloned in Mac Conkey agar+1% maltose+100ug penbritin/ml of phenotype, and called after χ 3938(798), χ 3961(UK-1) and χ 3774(SL1344), preceding two strain gene types all are △ crp-11 △ (zhc-1431 ∷ Tn10) pSD110 +, χ 3774(SL1344) genotype be △ crp-11pSD110 +
Bacterial strain χ 3774, χ 3938 and χ 3961 cultivate in the LB that 100ug penbritin/ml is arranged, and chain △ cya-12 and zid-6 ∷ Tn10 sudden change are introduced in the P22HTint transduction that is used in breeding among the χ 3711 respectively.The transduction mixture is coated on the flat board of Mac Conkey agar+1% maltose+100ug/ml penbritin+12.5ug tsiklomitsin/ml.Choose amicillin resistance (pSD110), tetracyclin resistance (zid-62 ∷ Tn10) and Mal -The clone of (△ cya), purifying on the flat board of Mac Conkey agar+1% maltose+100ug penbritin/ml+12.5ug tsiklomitsin/ml.The clone of purifying inoculates LB and is cultured to muddiness, checks the complete lipopolysaccharides (LPS) and the auxotroph proterties of bacterial classification.The bacterial strain called after χ 3978(798 that obtains), χ 3962(UK-1) and χ 3936(SL1344) preceding two strain bacterium genotype all are △ crp-11 △ (zhc-1431 ∷ Tn10) pSD 110 +△ cya-12 zid-62 ∷ Tn10, the latter's genotype is △ crp-11 pSD110 +△ cya-12 zid62 ∷ Tn10.χ 3936, χ 3978 and χ 3962 grow to muddiness in the nutrient solution of LB+100ug penbritin/ml+12.5ug tsiklomitsin/ml, be diluted to BSG at 1: 10, each 100ul nutrient solution is coated on the flat board that contains 5-butyl-pyridine-2-formic acid, and 37 ℃ are incubated 36 hours.5-butyl-pyridine of every strain bacterium-2-formic acid resistance clone is chosen and purifying on the FA flat board.The FA-resistance clone of purifying is inoculated among the LB, grows to muddy the losing of its Tn10 (sensitive tetracycline), complete lipopolysaccharides and the auxotroph proterties checked then.The pSD110 plasmid usually with spontaneous losing in the bacterial strain, causes the penbritin sensitivity in this process, but derives from except the bacterial strain of SL1344, and the elimination of its pSD110 relates to two steps.Last bacterial strain called after χ 4039(798), χ 3985(UK-1) and χ 3939(SL1344), the above two genotype are △ crp-11 △ (zhc-1431 ∷ Tn10) △ cya-12 △ (zid-62 ∷ Tn10), and the latter's genotype is △ crp-11 △ cya-12 △ (zid-62 ∷ Tn10) (table 1.B).
The genotype of non-toxic mutant bacterial strain and the stability of phenotype
The method of decision stabilization characteristics of genetics is seen described in the example one.The genotype and the phenotypic character of all △ cya △ crp sudden changes, motility except bacterium, all be stable, though there is the motility synthetic and bacterium of the flagellum of function to depend on the function of wild-type cya and crp gene, but be divided at cfs(composing type flagellum) be easy to be sieved to the sudden change of an inhibition in the gene, make the synthetic and bacterium motility of flagellum not rely on the function of cya and crp gene.In the △ of S.typhimurium cya and △ crp mutant strain, be easy to choose the different varient of motility in the strain construction process.Because the immunity to flagellar antigen may be a protectiveness, the different varient of the motility of all vaccine strains all is selected.
Antigenic synthesizing of S.typhimurium B group O confirmed from two aspects.One is to use the slide agglutination of antiserum(antisera) (Difco laboratory, Detroit, Michigan State), and two are to use Luria soft agar soverlay technique to measure its susceptibility to the P22HTint phage.
The double-mutant bacterial strain has only the single mutation bacterial strain of △ cya or △ crp identical to the fermentation of sugar with growing state on different carbon sources, and is listed as table 2.As desired, catabolic activity needs cAMP and cAMP receptor protein, the report that this is existing has delivered.
Choose 5-butyl-pyridine-2-methyl resistance but behind the bacterial strain to sensitive tetracycline, whether the frequency that ensuing each step makes up the sudden change all observed the bacterial strain with tetracyclin resistance or reverse mutation can be higher than the frequency that the wild-type parent bacterial strain to sensitive tetracycline occurs, and this situation is not all observed in each step.
Mutants which had is pathogenic to mouse
Pass through 10 about the preliminary data that the S.typhimurium mutants which had is pathogenic 8Individual mutant bacteria gives single mouse oral, and calculates pathogenicity rate and lethality rate and obtain.The data presentation of table 4 with S.typhimurium wild-type parent bacterial strain with the bacterial strain χ 3985 of △ cya-12 and △ crp-11 sudden change is arranged and pathogenicity rate and the lethality rate of χ 4039 after giving mouse oral.
(seeing Table 4)
Validity with the non-toxic mutant immunity
To the immunity of the full toxicity S.typhimurium bacterial strain of oral wild-type subsequently, the dosage of immunity can reach 10 of LD50 dosage with S.typhimurium △ cya △ crp mutant χ 3985 and χ 4039 inoculation back mouse for the data presentation of table 5 4Doubly.Under the situation of like this high dosage, a lot of mouse only are slight causes a disease and reduces with feed, and all keeps fit with χ 4039 mice immunized, and feed and growth are normally.
(seeing Table 5)
Example three
This example is described the separation of microorganism that a kind of crp of having gene and are adjacent the deletion mutantion of gene.This adjacent gene is pathogenic relevant with Salmonella.
Bacterial isolates Escherichiacoli and Salmonella typhimurium bacterial strain are listed in table 1A and B.Bacterial strain is preserved and is seen described in the example one.
The conventional cultivation of substratum used complex medium, and bacterial count and discriminating are seen described in the example one.
Transduction and losing with 5-butyl-pyridine-2-formic acid screening Tn10
Substratum and method see that example one is described.
The mensuration of zoogenetic infection and protective immunity
Pathogenic and the immunogenic mensuration of S.typhinurium bacterial strain sees that example one is described.
Have the separation of the S.typhimurium bacterial strain of △ crp-10 sudden change
Described in example one, have 1st/10th in the △ crp mutant that from χ 3605, is separated to, arginine auxotroph (because disappearance of argD gene) and halfcystine auxotroph (because disappearance of cysG group).The initial called after △ of sudden change crp-10 among the S.typhimurium SL1344 bacterial strain χ 3622, but because it is a halfcystine auxotroph, so present called after △ (crp-cysG).With 10 9After individual χ 3622 cells are given one group (five) BALB/c mouse are oral, mouse all keep fit state and not influence (table 3) fully.Even these mouse have obtained oral 10 8The high-level immunizing power of individual parent χ 3339 cells.
A series of bacterial strain is fabricated and is used for estimating independently each phenotype characteristic of χ 3622.The crp that has the clone +The plasmid pSD110(Schroeder and the Dobrogsz of gene and ampicillin resistance gene, J.Bacteriol, 167: 616-622 1986) be used to the △ crp sudden change on the complementary karyomit(e).Be used in the P22HTint transduction of breeding in S.typhimurium bacterial strain χ 3670 bacterial strains that have plasmid pSD110 and be incubated at χ 3622 among the LB.On the flat board of MacConkey agar+1% maltose+100ug penbritin/ml, elect.After 26 hours, amicillin resistance and Mal are arranged +The clone of phenotype is chosen and purifying on same flat board.This clone's called after χ 3706.After giving mouse oral with χ 3706, from the spleen of mouse, separate again.The bacterial strain that is separated to is called χ 3737.Other two strain is not arginine or the auxotrophic crp gene mutation body of halfcystine bacterial strain χ 3605(crp-773 ∷ Tn10) and χ 3623(△ crp-11) also by transduceing, be named as χ 3731 and χ 3774 respectively by after the PSD110 complementation.The S.typhimurium bacterial strain that has cysG and arg sudden change respectively is fabricated and called after χ 3910(cysG ∷ Tn10), χ 4063 and χ 4071(arg ∷ Tn10).
Pathogenic selected next the sudden change by introducing △ crp-10 of S.typhimurium bacterial strain of other two plant heights makes it reduction.χ 3761(UK-1) and 789 are the morbific infectivity bacterial strains that from dying horse and pig, are separated to respectively, it is to the LD of mouse 50S is about 1 * 10 5CFU.At first the phage P22HTint lysate of the high titre of preparation is beneficial to the transduction (seeing Table 1.B) of △ crp-10 and the transposon zhc-1431 ∷ Tn10 chain with it from S.typhimurium bacterial strain χ 3712.Phage splitting liquid is used for inherited character transduction then and enters wild-type receptor bacteria χ 3761 and 789.Filter out the tetracyclin resistance clone and identify its Mal -, Arg -And Cys -Phenotype, what obtain derives from 798 bacterial strain called after χ 4246, derives from χ 3761(UK-1) bacterial strain called after χ 4249(table 1).
Have wild-type crp by transduction +The plasmid pSD110 of gene enters χ 4246 and χ 4248, and the crp sudden change is by complementary.Be used in and have plasmid pSD110(table 1) bacterial strain χ 3670 in the P22HTint transduction of the breeding χ 4246 and the χ 4248 that in LB, cultivate.Flat board with MacConkey agar+1% maltose+100ug penbritin/ml+12.5 μ g tsiklomitsin/ul elects.After 26 hours, choose an amicillin resistance and Mal in each bacterial strain +The clone, called after χ 4247(798 respectively behind purifying on the same flat board) and χ 4262(UK-1), both are pSD110 at genotype +/ △ crp-10 zhc-1143 ∷ Tn10.
Pathogenic table 6 data presentation of S.typhimurium χ 3622, χ 3731, χ 3737, χ 3774, χ 3910, χ 4063 and χ 4071 pathogenicity rate and the lethality rate after giving mouse oral with S.typhimurium bacterial strain χ 3622, χ 3731, χ 3737, χ 3774, χ 3910, χ 4063 and χ 4071.3737 pairs of mouse totally nontoxics of bacterial strain χ use 10 4Times wild-type parent bacterial strain χ 3339LD 50Dosage, mouse is not showed the symptom of any disease yet in the whole 30 days observation period.As the contrast of this experiment, the crp-773 ∷ Tn10 of χ 3605 sudden change is wild-type Crp by the pSD110 complementation +After the phenotype (χ 3731), the infected back of mouse is dead, and about 1 * 10 5The dosage of CFU kills 4/5ths mouse.In order to test Cys -And Arg -The bacterial strain of phenotype pathogenic is with bacterial strain χ 3910(cysG ∷ Tn10), χ 4063(arg ∷ Tn10) and χ 4071(arg ∷ Tn10) give BALB/C mice oral.After giving mouse oral with χ 3910, the χ 4063 of identical or less dosage and χ 4071, mouse is killed.Therefore, with △ crp-cysG) the relevant nontoxicity of-10 sudden changes has more than is because the disappearance of crp gene, also just owing to the disappearance of argD or cysG gene.On the contrary, another one is that the pathogenic necessary gene one of S.typhimurium is positioned near the chromosomal region of crp gene.
(seeing Table 6)
The immune validity of bacterial strain χ 3622, χ 3737, χ 4247 and χ 4262
The bacterial strain of data presentation shown in the table 7 χ 3622, χ 3737, χ 4247 and χ 4262 can inducing mouses to oral or be equivalent to complete toxic belly inoculation wild-type S.typhimurium bacterium LD 50Dosage 10 4Immunity during the multiple dose bacterium.Any symptom that the mouse of all overdose wild-type parent bacterial strains is never showed in whole 30 days experimental sessions.Therefore △ (crp-cysG)-10 sudden change has lacked two genes at least.Make S.typhimurium totally nontoxic and have hyperimmunity.
(seeing Table 7)
Have the separation of the S.typhimurium bacterial strain of △ crp-14 sudden change
Because crp-773 ∷ Tn10 inaccurate cuts off the disappearance that has produced the gene beyond argD and the cysG gene region, other method is designed to determine position adjacent with the crp gene and the relevant gene with nontoxicity.20 χ 3910(cysG ∷ Tn10 independently) deletion mutant screens sensitive tetracycline and Mal on the flat board that contains 5-butyl-pyridine-2-formic acid -Phenotype.The χ 3910 mutant gene types of one of them anti-5-butyl-pyridine-2-formic acid are △ (crp-cysG) 14, and show the auxotroph of Histidine and halfcystine, but are not arginic auxotroph.This bacterium called after χ 3931.With the lysate transduction χ 3931 of P22HTint, so that introduce the plasmid pSD110 that has wild-type crpt gene to χ 3670.Amicillin resistance, Mal -Transduttant is chosen and in same substratum behind the purifying, the bacterium called after χ 3955 that obtains.
S.typhimurium's is pathogenic
PSD110 +/ △ (crp-cysG)-14 χ 3955 tables 7 show pathogenicity rate and the lethality rate behind the oral S.ty-phimurium χ 3955 of mouse.When oral about 10 9Behind the bacterium χ 3955 of CFU, mouse never showed any disease during whole 30 days, and 3955 pairs of mouse totally nontoxics of χ are described.
The immune validity of bacterial strain χ 3955
Table 7 shows that χ 3955 energy inducing mouses are to oral 10 4Times wild-type toxic strain LD 50The immunity of the bacterium of dosage.In 30 days experimental session, the mouse of overdose parent strain is never showed any illness.
χ 3622(△ (crp-cysG)-10) and χ 3737(pSD110 +△ (crp-cysG)-10) colony at digestive tube, GALT and spleen forms corresponding to wild type strain χ 3339, and S.ty-phimurium bacterial strain χ 3622 and χ 3737 are cultivated and give eight all big female BALB/C mice oral, see that example one is described.Oral 0.4 * 18 8CFU(χ 3622), 1.2 * 10 9CFU(χ 3737) or 1.1 * 10 9CFU(χ 3339) after one, three, five, seven day, mouse is killed respectively, selects three in every group of mouse at random, kills the sample of back collection organization.The polypropylene that long ileum of spleen, aggregated lymphatic follicles, 10cm and the small intestine of every mouse placed BSG in vitro is placed on ice with the homogenate of Brinkmann tissue homogenizer.Dilution or undiluted sample (100ul) are directly coated MacConkay agar+1% lactose+50ug Streptomycin sulphate/ml(χ 3339 and χ 3737) on dull and stereotyped and Mac Conkay agar+1% maltose+50ug Streptomycin sulphate/ml flat board (χ 3622), 37 ℃ are incubated 26 hours, measure every kind of in-house titre in each time limit, each the time limit the use of three mouse in every group sample calculate the geometrical mean of its titre.
Other reduction sudden change reduces bacterial strain greatly and forms ability at the colony of deep tissues among the bacterial strain χ 3622, and this is having crp +(pSD110 +) also still exist among the bacterial strain χ 3737 of phenotype.Therefore the gene that lacks because of △ (crp-cysG)-10 sudden change is named as cdt.The cdt of χ 3622 and χ 3737 -Phenotype is also confirmed to failing to observe mouse spleen enlargement phenomenon behind oral these the two kinds of bacterium of mouse, and △ cya sudden change (Curtiss and Kelly are arranged for the bacterial strain χ 3623 of the oral △ of the having crp-11 sudden change of mouse or other existing △ crp sudden change, 1987) behind the bacterial strain, but can observe the spleen enlargement phenomenon of mouse.Bacterial strain χ 3737 growth fraction χ 3622 are faster.Other reduction sudden change among the χ 3622, the same with the crp sudden change, do not reduce the growth velocity of bacterium.
Based on separation and the analysis to deletion mutantion, the order of gene is aryD crp cdt cysG on the S.typhimurium karyomit(e).
Clearly, △ (crp-cysG)-10 sudden change or △ (crp-cysG)-14 sudden change also are △ cdt sudden change certainly, can increase the security of attenuated vaccine bacterial strain alive, and not reduce its immunogenicity.This is to the infectivity Salmonella bacterial strain particularly important of those tool host specificities, for example Sityphi, S.porratyphiA(S.schottmnelleri), S.porratyphi B(S.hir-shfeldii), the equal infected person of S.paratyphiC(), S.choleraesuis(infected pigs), S.dublin(infects milk cow), S gallinarum and S.pullorum(all infect poultry), also the specific infectivity Salmonella bacterial strain of those nonhosts such as S.typhimurium and S.enteri-fidis are had the meaning of particularly important.
Example four
This example describe have influence cAMP contain to become influence the adjacent gene relevant that bacterial strain forms at animal deep tissues colony with the structure and of the avirulent strains of the deletion mutantion that utilizes with Salmonella toxicity and stablize the phenotype that has two bacterial strains that suddenly change, the characteristics of totally nontoxic, high immunogenicity.
Bacterial isolates Escherichia coli and Salmonella typhimunium bacterial strain are listed in table 1A and B.Bacterial strain is preserved and is seen that example one is described.
The conventional cultivation of substratum used complex medium.Bacterial count and discriminating see that example one is described.
Transduction and screen Tn10 with 5-butyl-pyridine-2-formic acid and lose
Substratum and method see that example one is described.
Have △ cya-12 and △ (crp-cysG)-10 deletion mutantion the S.typhimurium bacterial strain the structure best efficiency vaccine strains may by have strong cloning ability and infectious high toxicity bacterial strain the reduction and obtain.Highly pathogenic S.typhimurium wild type strain is as SL1344(χ 3339), UK-1(χ 3761) and 798 choice criteria see as described in the example 2.
With with Curtiss and celly(1987) method of described similar classical genetics, to the toxic strain SL1344 of wild-type, 798 and UK-1 carry out genetic modification.With the Tn10(tetracyclin resistance of encoding) insert near SL1344 △ cya-12 or △ (crp-cysG)-10 mutator gene, enter high toxicity bacterial strain UK-1(χ 3761 by the transduction of P22HTint proterties that these are chain), 798 and SL1344(χ 3339) bacterial strain, select tetracyclin resistance and Mal -Phenotype, the deletion mutantion of crp and cya gene are separated in S.typhimurium SL1344 and are identified out (seeing that example one is described).The zid-62 ∷ Tn10 chain with the chain zhc-1431 ∷ Tn10 of △ (crp-cysG) 10 and △ cya-12 is used in experiment.Two kinds are inserted sudden change and are not influenced the pathogenic of S.typhimurium separately.
At first respectively the bacterial strain χ 3712 that has △ (crp-cysG)-10 and zhc-1431 ∷ Tn10 sudden change and have △ cya-12 and the bacterial strain χ 3711 of zid-62 ∷ Tn10 sudden change in the phage P22HTint lysate of the high titre of preparation.The lysate that the obtains inherited character that is used for transduceing enters wild type strain χ 3339,798 and χ 3761.
At bacterial strain χ 3712(△ (crp-cysG)-10zhc-1431 ∷ Tn10) in the P22HTint of breeding be used for the toxic strain of transduceing, filter out tetracyclin resistance and Mal -Proterties.Phage and infectation of bacteria lysate 37 ℃ the insulation 20 minutes after, get the 100ml sample and coat Mac Conkey agar (Difco laboratory, the Detroit, the Michigan State)+1% on the flat board of maltose (ultimate density)+12.5ug tsiklomitsin/ml, 37 ℃ of insulations were chosen and are had tetracyclin resistance and Mal after 26 hours -The clone of phenotype and on same plane purifying.What obtain derives from 798 bacterial strain called after χ 3777, derives from the bacterial strain called after χ 3779 of ulc-1.Bacterial strain χ 3712, χ 3777 and χ 3779 genotype are △ (crp-cysG)-10 zhc-1431 ∷ Tn10(table 1.B).χ 3779 and χ 3777 are after the LB liquid culture of band 12.5ug tsiklomitsin/ml, go into gelatin buffer salt solution (BSG) with 1: 10 dilution proportion respectively, respectively getting 100ul coats and contains flat board (Maloy and the Nunn that is dissolved in 5-butyl-pyridine-2-formic acid (FA), 1981) on, 37 ℃ are incubated 36 hours approximately.The clone who chooses the FA resistance enters 0.5mlBSG purifying on the FA substratum.Clone behind the purifying inoculates LB liquid and grows to muddiness at 37 ℃, checks existence of losing (sensitive tetracycline), complete lipopolysaccharides and the auxotrophy of its Tn10.New bacterial strain called after χ 3784(UK-1) and χ 3806(798), both genotype are △ (crp-cysG)-10 △ (zhc-1431 ∷ Tn10).The initial separation of χ 3622(SL1344 △ (crp-cysG)-10 is seen example one described (table 1B).
Because Cya -And Crp -Identical (the Mal of plain type of mutant -, Stl -, Mtl -Deng), have clone's crp +Gene and ammonia benzyl mould become the plasmid pSD110(Schroeder and the Dobrogosz of resistant gene, J.Bateriol, 167: 616-622,1986) be used to the △ crp sudden change on the of short duration complementary karyomit(e), to identify the △ cya sudden change of introducing by transduction.Be used among the S.typhimurium bacterial strain χ 3670 that has pSD110 χ 3622, χ 3784 and χ 3806 that the P22HTint transduction of breeding is cultivated in LB liquid.Selecting flat board is Mac Conkey agar+1% maltose+100ug penbritin/ml.After 26 hours, each bacterial strain is chosen a tool amicillin resistance and Mal +The clone of phenotype behind purifying on the identical flat board, is named as χ 3901(798 respectively), χ 3945(UK-1) and χ 3706, the above two genotype are △ (crp-cysG)-10 △ (zhc-1431 ∷ Tn10) pSD110 +, the latter's genotype is △ (crp-cysG)-10pSD110 +
In order to introduce chain △ cya-12 and zid-62 ∷ Tn10, the P22HTint transduction that is used among the χ 3711 breeding is grown in bacterial strain χ 3706, χ 3901 and the χ 3945 in the LB liquid that contains 100ug penbritin/ml.The transduction mixture is coated on the flat board of Mac Conkoy agar+1% maltose+100ug penbritin/ml+12.5ug tsiklomitsin/ml.Choose tool amicillin resistance (pSD110 +), tetracyclin resistance (zid-62 ∷ Tn10) and Mal -The clone of (△ cya) phenotype and on same flat board purifying, the clone behind the purifying is seeded in the LB liquid and grows to muddiness, checks its complete lipopolysaccharides and auxotroph.The bacterial strain called after χ 3902(798 that obtains), χ 3956(UK-1) and χ 3722(SL1344), the above two genotype are △ (crp-cysG)-10 △ (zhc-1431 ∷ Tn10) pSD 110 +△ cya-12zid-62 ∷ Tn10, the latter's genotype is △ (crp-cysG)-10pSD110 +△ cya-12zid-62 ∷ Tn10.χ 3722, χ 3902 and χ 3956 grow to muddiness in the LB liquid that contains 100ug penbritin/ml and 12.5ug tsiklomitsin/ml after, to be diluted to BSG at 1: 10, get 100ul sample coating 5-butyl-pyridine-2-formic acid flat board, 37 ℃ are incubated about 36 hours, choose resistance clone purifying on the FA flat board.Clone behind the purifying is inoculated in the LB liquid and grows to muddiness, check that its Tn10 loses (sensitive tetracycline), in this process of complete lipopolysaccharides and auxotroph, pSD110 Chang Zifa loses and causes bacterial strain to the penbritin sensitivity, but derive from except the bacterial strain of SL1344 and UK-1, the elimination of pSD110 needs two steps in these bacterial strains.χ 4038(798) and χ 3724 last bacterial strain called after χ 3958(UK-1).(SL1344) the above two genotype are △ (crp-cysG)-10 △ (zhc-1431 ∷ Tn10) △ cya-12 △ (zid-62 ∷ Tn10), and the latter's genotype is △ (crp-cysG)-10 △ cya-12 △ (zid-62 ∷ Tn10) (table 1.B).
The genotype of non-toxic mutant and phenotypic stability
Measure the method for stabilization characteristics of genetics and see that example one is described.The genotype and the phenotypic character of all △ cya △ crp sudden changes, motility except bacterium, complete stabilities all, though the movability of bacterium and the synthetic mould function that depends on the cya and the crp gene of wild-type of the flagellum of function is arranged is synthetic at cfs(composing type flagellum) be easy to select to obtain to make that flagellum is synthetic does not rely on the mutation inhibiting of crp and cya gene function with the bacterium motility in the gene.In the strain construction process, in S.typhimurium △ cya △ crp bacterial strain, be easy to choose the varient of different motilities.Because the immunity to flagellar antigen may be a protectiveness, the varient that different motilities are arranged of all vaccine strains all is selected.
Antigenic synthesizing of S.typhimurium B group O confirmed from two aspects.One is to use the slide agglutination of antiserum(antisera) (Difco laboratory, Detroit, Michigan State), two susceptibility to P22HTint that are to use Luria soft agar soverlay technique to measure.
Two mutant strains are identical with growing state on different carbon sources with the single mutation bacterial strain that has only △ cya or △ crp to the fermentation of sugar, see Table 2 listed.As expection, catabolic active cAMP and cAMP receptor protein of needing, this existing report of publishing.
Choose 5-butyl-pyridine-2 formic acid resistance but behind the bacterial strain to sensitive tetracycline, whether the frequency that ensuing each step makes up the sudden change of all having observed the bacterial strain with tetracyclin resistance or reverse mutation can be higher than the frequency that the wild-type parent bacterial strain to sensitive tetracycline occurs.This situation is not all observed in each step.
Example five
The structure of the nontoxic Salmonella bacterial strain of expressing human LDH-C
A pYA3042
Plasmid pYA810(Fig. 3) is the asd that can in the nontoxic Salmonella vaccine strains that △ cya △ crp △ asd sudden change is arranged, use +Cloning vector.It derives from plasmid pYA292(Fig. 2).Multiple clone site on the pYA810 provides several approach for clone's the expression of DNA sequence under the control of the trc of constitutive expression promotor.PKA3042(Fig. 5 in the recombinant plasmid that use pYA810 makes up) be a representative.Have the special LDH-C order of human sperm on it, under the control of the trc of pYA810 promotor, be expressed as fusion rotein.From the SmaI-Hind III site that the SmaI-Hind III fragment of plasmid pHUM-LDH-C cutting-out is inserted pYA810.Above-mentioned linker transformed has pYA232(Fig. 6) bacterial strain χ 6212(△ EargF-lacZYA) U169 glnV44d λ-ψ 80d/lacZ △ M15 gyrA96recAl relAl endAl △ zhf-z ∷ Tn10hsdR17) obtain plasmid pYA3042, plasmid pYA232 has the replicon of pSC101, and LacI is provided qThing is met in resistance.Thereby Westem inhales the mark analysis revealed makes the trc promotor not be subjected to IacI containing inductor IPTG when bacteria growing qThe following time of condition of suppressing, two in E.coli host have that the segmental clone of the correct insertion of size and Orientation can produce can be with the protein of the polyclone precursor reactant of mouse LDH-C.One of them clone, called after pYA3042(Fig. 5), directly change band △ cya △ crp △ asd sudden change S.typhimurium bacterial strain χ 3987 over to electroporation method.Westem inhales that seal analysis determined once more can be with the proteinic generation of the antibody response of polyclonal anti-mouse LDH-C.There is this immunoreactive protein in having the bacterial strain χ 3987 of pYA810, can not detect.
B.pYA3054
Plasmid pKKHC4(Fig. 7, Levan and Goldberg 1991) have the open reading frame of 966 base pairs of the LDH-C of coding people sperm specificity.With rabbit anti-serum and mouse LDH-C 4The monoclonal antibody of (Millan etc., 1987) screening from people's testis cD-NA expressing gene storehouse that figure λ gt11 makes up obtains the cDNA clone of LDH-C.Be connected after downcutting with Hinc II and DraI and add the XmaI joint, this open reading frame is cloned into pKK233-3(Pharmacia) the XmaI site.
EcoRI-Hind III fragment with the long 1.1Kb that obtains having the LDH-C gene behind the partially digested and Hind III complete degestion pKKHC4 of EcoRI.This fragment cloning places under the control of trc promotor (Fig. 8) the LDH-C expression of gene between the EcoRI Hind III site of pYA810.To connect mixture with electroporation method Transformed E .coli host χ 6212(pYA232, produce LacI qThe LDH-C clone pYA3054 that (Fig. 6)) obtains can produce the band of a treaty 35KD.Induce the activated LDH-C tetramer that the back produces can be with IDTG with rabbit to the LDH-C(mouse) the antiserum(antisera) reaction, the tetramer is measured by the test of the LDH on the non-denaturing polyacrylamide seed lac.This has the LDH-C clone pYA3054 of function directly to be changed over to χ 3987(△ cya △ crp △ asd with electroporation method), obtain the S.typhimurium vaccine strains of an energy constitutive expression LDH-C.
Before with this bacterial strain immune mouse, with the phenotype of this bacterial strain, growth velocity and plasmid stability are with the vaccine strains χ 3987(pYA810 that only has carrier) contrast.Test χ 3987(pYA810) and χ 3987(pYA3054) following characteristics: phage P22 susceptibility (smooth lipopolysaccharides), the growth on defined medium (prototroph), unfermentable maltose (△ cya △ crpMal -Phenotype), contained plasmid (90Kb toxicity plasmid and 44kbpYA3054) and sensitive tetracycline (no lacI qPlasmid pYA232) measures the correct (P22 of phenotype s, Prot, Mal -, Tc s) pYA810 and pYA3054 electroporation transformant in the growth velocity on the Lenox substratum and containing cultivated for 60 generations in the Lenox substratum of diaminopimelic acid (DAP50ug/ml) after the structure and the isolating stability of plasmid.Under airproof condition, in containing the Lenox substratum of DAD, cultivate about 60 generations after, with agarose gel electrophoresis with the LDH proteins gel electrophoresis mensuration plasmid pYA810 of function and structure and the segregational stability of pYA3054 are arranged.These tests show, 10 strain χ 3987(pYA3054) in 10 strains plasmid and the active LDH-C of 4.4kb are all arranged, from LA +DAP(50ug/ml) duplicate the χ 3987(pYA3054 of LA substratum on the flat board) and χ 3987(pYA810) in have 99.2% and 99.3% to contain Asd respectively +Plasmid.Inference χ 3987(pYA3054 thus) can be used to inoculate mouse.
PYA3054 can be introduced the vaccine strains that other has △ cya △ crp △ asd sudden change now, for example above-mentioned S.typhi bacterial strain makes the host specificity of their acquisitions to the people.In contrast, also should be structured in the clone of expressing human LDH-C on higher or the lower level, these LDH-C can be used as the component on periplasm or surface, and perhaps the labile toxin (LT) with enterotoxigenic E.coli coding forms fusion rotein.
Example six
The structure of the recombinant avirulent salmonella of coexpression human LDH-C and E.coli heat-labile toxin B-subunit (LT-B).
A.pYA3095 and pYA3097
Utilize the LT-B carrier pYA3048(described as follows), make up two coexpression recombinant plasmids and expressed LDH-C and LT-B.LT-B B subunit is as a kind of oral adjuvant.And can combine with agarose and GM-1 Sphingolipids,sialo, be convenient to protein purification like this with its fusion.
PYA3048(Fig. 9 A) derived from pYA810, comprises the LT-B sequence, and do not hold at LT-B and to have multiple cloning site and add two translation stop codon (Fig. 9 B) that are in different reading frames.
PYA3095 makes up from pKKHC4 and pYA3048, and as shown in figure 10, the result has the LDH-C gene of tac promotor from pKKHC4, is in the downstream of the LT-B gene of band trc promotor.Though it seems that originally this clone can not hinder and comprises lacI qIntestinal bacteria (E.coli) the host χ 6212(pYA232 of repressor (carry by pYA232, see Fig. 6)) growth, but induce through 0.5mMIPTG, can be observed bad growth.When this clone introduces χ 3987 through electroporation, also observe bad growth.When whether detect χ 3987(pYA3095 with the Western hybrid method) produced LT-B and LDH-C, only detect low-level LT-B.
PYA3097 is another plasmid that makes up, as shown in figure 10.This plasmid is to constitute by Bg1 II site among the insertion of the 1.6kbBg1 II fragment on the plasmid pYA3054 of the LDH-C coding region that will have trc promotor and the 5s T1T2 terminator pYA3048.Being thus connected the plasmid pYA3097 that forms causes the LDH-C that has the trc promotor to be in counter-clockwise direction.Seem that this clone can be unharmful to the host after IPTG induces.After χ 3987 was advanced in commentaries on classics, host's growth velocity slowed down, and, in the segregational stability test, just there is recombination event to take place, thereby eliminated the LDH-C part among the clone.These LDH -Bacterium becomes in colony and preponderates.Activity according to 10 strain isolates detects and the analysis of plasmid size, and they account for more than 90% of colony.
B. other LDH-C/LT-B recombinant plasmids
Recombinant plasmid with single promotor can followingly be constructed.In order to produce LT-B and the LDH-C operon fusant that is under the control of trc promotor, can construct a synthetic primer, comprise a ribosome bind site (RBS), be positioned at before the LDH-C initiator codon 10-15bp.Utilize polymerase chain reaction to produce the LDH-C coding region then, have suitable BamHI and PstI site so that be cloned among the pYA3048.
The LDH-C/LT-B fusant also can make up by mode as shown in figure 11.Separation is processed into tack from the 0.9kb of pYA3054 or pKKHC4 EcoRI(through mung-bean nuclease)-PstI LDH-C coding region and MluI(Klenow mend flat)-pYA3048 or pYA3082 carrier that the pstI enzyme is cut are connected, thus generation LT-B-LDH-C protein blend zygote.
Example seven
Express the structure of the recombinant avirulent salmonella of mouse LDH-C
Be used to the one section EcoRI fragment that comprises the LDH-C gene, made up a mouse LDH-C clone from mouse cDNA clone.This fragment cloning is to pBS(stratagene, La Jolla, California), produce plasmid pBS mLDH.Though the sequence of mouse LDH-C is known, the LDH-C of being cloned into carrier pBS EcoRI site is known seldom.Can keep the initial and ribosome bind site of LDH-C when supposing the clone, or make initiator codon be positioned at the ribosome bind site that appropriate position is utilized a portion, thereby by separating the EcoRI fragment of the about 1.3kb of a size among the pB-SmLDH, be connected to pYA810 EcoRI site, and attempt to clone.With pvu II and Hind III enzymolysis, LDH-C inserts the correct directed clone of fragment like this, will produce the fragment of the about 1.0kb of a size.Yet, can not detect the LDH of function.The undetermined character of these mouse LDH-C clone's 5 ' one ends makes and need design a primer on existing sequence data basis, directly clones mouse LDH-C to pYA810, produces the LDH-C that function is arranged.
Example eight
The purifying of people LDH-C
LDH-C can purifying, and is used in the ELISA(enzyme-linked immunosorbent assay) in as envelope antigen, detect by the immune response of immunizator.LDH-C can separate from arbitrary recombinant expression by the method for standard.Yet some LDH-C that comprises the plasmid generation of the LT-B that merges with LDH-C still merges with LT-B.Therefore, the pMAL carrier (Figure 12) that utilizes New Eng-land Biolabs to provide has made up another kind of plasmid, makes to separate the LDH-C that does not have LT-B.This system comprises that with relevant albumen and maltose binding protein fusion, then purified fusion protein on the amylose starch post is used free maltose wash-out again.
This recombinant plasmid result produces a large amount of MBP-LDH-C fusion roteins, and translation product and MBP itself greatly, and all these is combined on the amylose starch post.The MBP-LDH-C fusion rotein that this recombinant plasmid produces has a factor Xa(Factor Xa near LDH-C is initial) site (factor Xa recognition sequence different bright-paddy-Gan-essence, do not have this sequence among the people LDH-C).Product cuts with Xa.A kind of extra three amino acid whose LDH-C that have have been produced with this proteolytic enzyme cutting result.Enzymolysis product separates the LDH-C and the LDH-C portion of product that obtain low-level amount then by another amylose starch post.Therefore, MBP-LDH-C fusion rotein mixture just is used as envelope antigen and is used for ELISA.Fusion rotein is attached to the ELISA flat board, and the fusion rotein of concentration 10 mcg/ml can be to the rabbit LDH-C(mouse of 1: 1000 times of dilution) serum provides optimum response; And can not detect 3987(pYA810 with mouse α χ) cross reaction of serum.Therefore, this fusion rotein is applicable to ELISA.
Can make up another kind of C-end LDH-C-6XHis fusion rotein recombinant plasmid, sharp system is separated total length LDH-C with QIAGEN nickel coupling system.
Example nine
Animal immune
The recombinant plasmid of above-mentioned arbitrary expression LDH-C all can be introduced other suitable non-toxic microbes, as △ cya △ crp △ asd S.typhimurium bacterial strain.This bacterial strain is derived from one can be at enteron aisle GALT particularly, effectively the infectious bacterial strain of accumulative height.The domestication of height infectivity bacterial strain from bringing out immunology, causes having produced a kind of super vaccine.χ 4072 and χ 3987 are exactly the example of this bacterial strain.In these bacterial strains, the stability of recombinant plasmid can be by measuring microbial culture being added with and not adding in the substratum of diaminopimelic acid (DAP); And can measure the amount of LDH-C, stability (by the pulse-chase method) and the position in bacterial cell.What deserves to be mentioned is that the fusion rotein that merges with LT-B may be to be transported in the pericentral siphon by cytoplasmic membrane.The recon of choosing can be tested, and whether has the molecular configuration of expection with the check plasmid, and by polyacrylamide gel electrophoresis, dyeing detects LDH activity (Goldberg 1964) and comes the analysing protein extract.Anti-LDH antibody can be used for Westen engram analysis (Towbin etc. 1979), and this is important when enzymic activity does not detect.
At last, bacterial strain grows to logarithmic phase in LB liquid, and centrifugation concentrates in the buffer salt solution that is added with gelatin, is used for oral.
A, mouse immuning test
Utilize the χ 3987(pYA3054 of expressing human LDH-C), only to have the χ 3987(pYA810 of carrier) be contrast, the LDH-C structure is tested in mouse.Experimental result has disclosed the stability of the clustering capability and the structure of expressing the LDH-C vaccine strains in vivo.The first strain mouse of selecting is C57B16.It is reported that this histocompatibility group's mouse is good to the LT-B reaction, and a good model so just is provided, and comes comparison LT-B and LDH-C recombinant plasmid.Freezing original seed χ 3987(pYA810) and χ 3987(pYA3054) (phenotype correct and once be used for stability test) be used as the bacterium inoculation 2-3 milliliter Lenox substratum that sets out, 37 ℃, stuffiness is cultivated.Diluted culture in warm Lenox substratum by 1: 20 then, aerated culture to optical density(OD) is about A 6001.0 centrifugation cell is suspended in the buffering salt gelatin solution again.According to standard method, age in 12 8 of oral vaccinations week female mice C57B16, inoculum size is 1.2 * 10 9Colony-forming unit (CFU) χ 3987(pYA810), simultaneously, other 12 mouse are with 9.6 * 10 8The LDH-C(people of CFU) inoculation vaccine strains χ 3987(pYA3054).Behind the primary vaccination 10 days, get 6 mouse for every group, be used for immune Research,, give 1.5 * 10 more respectively according to the primary vaccination step 9The χ 3987(pYA810 of CFU) and 8.6 * 10 8The χ 3987(pYA3054 of CFU) inoculum size.The clustering capability of two kinds of bacterial strains was respectively got 3 mouse respectively and is detected from the primary vaccination group at the 7th day and the 14th day.
Be shown in Figure 13 and table 8 from the CFU sum of peyer's patches (pp) and spleen (sp) recovery, the result shows χ 3987(pYA3054) quantity that exists few slightly (though the χ 3987(pYA3054 that has the 7th day mouse) do not have cluster, do not included).These mouse are healthy always in research process, and still, after dissecting these mouse on the 14th day, they have the spleen and the liver of increase, and it is required that the prompting dosage of giving may be higher than this strain mouse.By respectively get 5 from 3 the 14th day different isolating bacterium colonies of mouse spleen, test has the LDH activity of function, measures the structural stability of pYA3054 in the body.The separation bacterium colony of all 15 detections according to LDH activity gels method, has all produced the LDH-C that function is arranged, and shows in the colony that arrives spleen more than 93% at oral immunity after 14 days.Expressing LDH-C.Be used for follow-up analysis simultaneously one group of mouse, another group mouse has been carried out the immune response detection by collecting serum.Serum was collected in the vaginal secretions in turn weekly respectively from menses and glandula from elementary immunity back in the 4th thoughtful the 8th week.Put into male mice C57B16 after the 8th week to female mouse (1 hero/2 are female).
The immune response of two groups of mouse is reacted by ELISA detection serum IgG and IgA and is measured, and the index that the mouse fertility is reduced detects.
B. rabbit immunity test
Great majority are relevant induces the immunoreactive research of general secretor type to check glandula, the intestines washes, and in the tears, the sIgA titre in suckling sometimes.Having only very limited work is the generation of sIgA in the relevant reproductive tract, is secreted into result among the GALT as antigen.Work for this reason, 5 does have been carried out immunity,, studied its secretor type and the immune response of body fluid type according to the method for top research mouse.In addition, collect vaginal secretions, be used for the anti-LDH-C sIgA of quantitative analysis by lavation.These experiment special values are that rabbit is outbreed, and immunoreactive some difference can expect, and exists significant difference if depend on the immune response to LDH-C of histocompatibility and Ir type.If run into this variability, a kind of antigenic vaccine of several sperm-specifics of expressing will be useful.
Example ten
1. express the structure of the recombinant avirulent salmonella of SP-10
A. site-directed mutagenesis is modified the pYA810 carrier
λ gtll clone SP-10-5 and SP-10-10(Wright etc., 1989) available EcoRI excises from carrier λ gtll.But, with the SP-10-5 sequence clone to the pYA810EcoRI site, can produce the fusant that reading frame is not inconsistent, and make its amino acid be positioned at fusion rotein N-end, and lower the secernment efficiency of protein probably by E.coli and S.typhimurium cytoplasmic membrane.Produce on pYA810-NcoI site (CCATGG) by site-directed mutagenesis like this, the result makes the sequence C CATGCCG GAA TTC of coded amino acid first sulphur-dried meat-paddy-phenylpropyl alcohol become the sequence C C ATG GCG GAA TTC of coding first sulphur-third-paddy-phenylpropyl alcohol.This carrier use has been to produce a useful cloning site, (because pYA810 is provided with the Ncol site).The correct nucleotide sequence of the pYA810 of sudden change keeps the EcoRI site by the existence of verifying new NcoI site simultaneously by the order-checking decision.
B. the structure that has the carrier of SP-10N-terminal sequence
Building process is illustrated in Figure 14.The pYA810-NcoI carrier is cut with the EcoRI enzyme, will cut from SP-10-5(Fig. 1 of λ gtll clone) 635bp EcoRI fragment (promptly from base pair 62 to 696) insert by connecting.This recombinant plasmid changes host E.coli χ 6212(recA hsdR △ asd over to by conversion or electroporation).This host has plasmid-encoded lacI qGene, the expression of any sequence of trc promotor control as a result depends on the existence of inductor IPTG.By cutting once on the SmaI(carrier, see Fig. 4) and the Xho II (point of contact is positioned at from 635bp SP-10 sequence 5 ' one end 40bp places, see Fig. 1) and SmaI and BclI(point of contact be positioned at SP-10 encoding sequence 3 ' one end 45bp places, see Fig. 1) restriction analysis, identify the recombinant clone of the SP-10-5 sequence that has correct orientation.Selected a clone who has correct orientation, analysis revealed, produce can with anti--protein that SP-10 antibody reacts (polymerization is a terminator codon after advancing 8 amino acid of polypeptide, sees Figure 15).
With this plasmid DNA then with the complete enzymolysis of NcoI, and with EcoRI part enzymolysis (this can cause the excision of the SP-10-5 sequence inserted sometimes, but sometimes not can, see Figure 10).Add the good linker of following hybridization in mixed solution:
5'GATGAACCAG3'
3'TTGGTCTTAA5'
This linker is connected with the carrier of incision, and the result inserts arginine and glutamine codon behind ATG methionine(Met) codon.(make the N-end become more alkaline by replacing AAG Methionin codon, whether can improve secretion with AAC arginine codon or CAG glutamine codon.Under arbitrary situation, EcoRI has recovered in the site.) this recombinant plasmid still just should not synthesize the SP-10 proteantigen, this will be proved.The plasmid of Chan Shenging is with EcoRI part enzymolysis like this, and outstanding the processing with nuclease of 5 ' strand removed, and then carries out tack and connects, and lack -4bp.This recombinant plasmid is inhaled the screening of seal method by the bacterium colony immunity and is produced the antigenic transformant of SP-10 by transforming or electroporation introducing E.coli bacterial strain χ 6212.Positive colony is analyzed whether have correct different restriction endonuclease sites (seeing Fig. 1 and 4).Compare with the SP-10 aminoacid sequence, unique difference is inserted a glutamine at the 3rd amino acid place exactly.At C one end, there is not terminator codon immediately on the carrier, this protein of Chan Shenging has 29 extra amino acid like this, because first transcription termination signal in reading frame is arranged in pYA810 carrier rrnB sequence.(Here it is to recombinant vectors pYASP-10-5 +The basis of "+" in the name.)
Carried out preparatory experiment and detected and add and cell growth condition when not adding IPTG, and whether when having DAP recombinant plasmid can selectivity lose.Also coldly oozed shock fractional separation raji cell assay Raji whether SP-10 albumen has been transferred to periplasmic space effectively by using.Whether on this basis, it is effective to have measured the unnecessary aminoacid sequence of C-terminal, harmful or neutral.If deleterious, will cut pYASP-10-5 with the EcoRI enzyme +DNA, insert following linker:
5'AATTCTGACGATT3'
3'GACTGCTAATTAA5'
This will cause connecting the TAA terminator codon behind the password of one one asparagus fern one asparagus fern of encoding amino acid sequence asparagus fern acyl.The plasmid called after pYASP-10-5ter that produces like this.
C. C-is held the SP-10 sequence to insert pYASP-10-5 +
Another kind of stop translation, the method for eliminating non-SP-10C one terminal amino acid sequence is to insert this sequence of EcoRI-XbaI SP-10 sequence (about 162bp) to derive from λ gtll and clone SP-10-10(and see Fig. 1).The building process of this generation pYASP-10ter is shown in Figure 16.Separate this fragment (EcoRI-XbaI), with the pYASP-10-5 of itself and EcoRI and the complete enzymolysis of SmaI +DNA connects.Afterwards, add archaeal dna polymerase Klenow fragment and mend flat xbaI otch, be connected with tack SmaI is terminal then.Produced total length SP-10 like this, its C one end Ile(is different bright) a then TAG termination codon of codon.This protein of synthetic in the presence of IPTG (except having an extra glutamine as the 3rd amino acid) should be the same with SP-10 albumen.Therefore this protein has 266 amino acid.
D. the structure of the SP-10 that merges with LT-B
Plasmid pYASP-10ter(Figure 16) complete with the AlwI enzymolysis, enzyme is cut carrier and is mended flat AlwI site with Klenow, then uses the PstI enzymolysis, reclaims 769bp fragment (see figure 1).LT-B fusion vector pYA3048(Fig. 9 A) with ApaLT enzymolysis (Fig. 9 B), flat with the Klenow benefit, then with the complete enzymolysis of PstI.These two kinds of molecules are coupled together, and recombinant molecule is introduced E.coli χ 6212 through electroporation.This plasmid pYALT-B-SP-10 building process is shown in Figure 11.LT-B-SP-10 fusion rotein synthetic will depend on IPTG and induce, and with the antiserum(antisera) of anti-LT-B and SP-10, carry out the analysis of Western blotting, measure it and synthesize.Also by carrying out the cell grade Separation Research and measure fusion product and whether be present in all spaces with cold shock method and the Western engram analysis of oozing.If measure fusion rotein again and in pericentral siphon, whether form pentamer.The LT-B pentamer in 0.1%SDS 60 ℃ be stable, behind the sds page electricity arteries and veins, also with anti-LT-B monomeric igg reaction.In the presence of 0.1%SDS, pentamer LT-B is 70 ℃ and above depolymerization, produces monomer molecule, at this moment can with anti-LT-B subunit antiserum(antisera) kickback.Like this, whether test immunogenicity announcement pentamer forms after the treatment of different temperature.
E. the characteristic research of recombinant clone
To pYASP-10-5 +, pYASP-10-5ter, pYASP-10ter and pYALT-B-SP-10 have carried out comparison test widely at E.coli χ 6212 with in S.typhimurium △ cya △ crp △ asd bacterial strain χ 4072 and χ 3987.Checked to add and the growth velocity when not adding IPTG, with ELISA or quantitatively Western engram analysis (using a molecular dynamics photodensitometer) measured the quantitative level of expression.The gene product location of expressing is by measuring with the cold shock cell fractionation method that oozes.Significant period of time is measured whether any genetic instability is arranged in the culture growth.SP-10 nucleotide sequence (Fig. 1) is carried out Computer Analysis disclosed numerous forward repetitions and the oppositely living complex sequences of part.Forward repeats to cause the increase or the minimizing of encoding sequence length, as the result of plasmid replication process generation of neutrons interchromosomal recombination.There is this class genetic instability if disclose,, and can not uses the nontoxic Salmonella of recA because recA further makes the Salmonella attenuation.But, can use the mutant of band recF sudden change, because recF stops between plasmid or the reorganization (James etc., 1982) in the plasmid and may the toxicity of Salmonella not had retroaction and make vaccine strains highly attenuated.
By cultivating for 50 to 100 generations, when having measured existence or not had DAP, whether plasmid still keeps.Also utilize pulse-chase to measure proteinic stability.At last, for the LT-B-SP-10 fusion rotein, itself and the combining of GM-1 Sphingolipids,sialo and/or agarose have been measured, as a kind of step of this fusion rotein of purifying.
2. the Asd that has N-end SP-10 sequence +The structure of carrier
Made up a kind of Asd +Carrier pYA3098, adjacent Shine-Dalgarno sequence downstream, a NcoI site (Figure 20) on it.This carrier is very convenient in making up a series of antigenic structures of all or part of SP-10 that have.Utilize 163 amino acid whose SP-10 sequences of pcr amplification decision N-end, this sequence is from adjacent SP-10 signal sequence cleavage site glycosides propylhomoserin (Fig. 1) before.The synthetic oligonucleotide is used as a fragment synthetic primer.Its 5 ' end be oligomer 5 '-GACCATGGGAACATCAAGTCAG-3 '.Except initial 7 Nucleotide, comprising the NcoI site, outside, all the other all are present in the SP-10 encoding sequence.3 '-terminal oligonucleotide is 3 '-GTCCACGTGGT-TAAAGTTCGCTTAAGGG-5 '.This oligonucleotide comprises an EcoRI site and holds in coding region C-.In order to prevent synthetic in the terminal non-SP-10 polypeptides derived of SP-10 sequence, (Figure 20) inserts one section oligonucleotide between pYA3098EcoRI and BamHI site, at each reading frame terminator codon to be arranged all.Synthesized oligonucleotide 5 '-TTAAGTAGGTAAATAG and complementary product thereof, 3 ' CATCCATTTATCCTAG5 '.After the hybridization, this two sequence is inserted into the pYA3098 that uses EcoRI and BamHI enzymolysis in advance.This can cause inserting TAG, TAA and TAG terminator codon at three kinds of reading frames in any.This novel vector is called pYA3098stop.SP-10 encoding sequence NcoI and the complete enzymolysis of EcoRI with pcr amplification are connected with the pYA3098stop of E.coRI enzymolysis with NcoI.Recombinant vectors is introduced E.coli χ 6212(DH52 with electroporation derive, have and comprise lacI qThe plasmid pYA232 of gene).In the pYA3098stop carrier (Figure 20), therefore the segmental expression of SP-10 of trc promotor control will depend on the adding of IPTG.One 167 amino acid whose polypeptide of the special coding of the structure of Chan Shenging like this.Can use other clone strategies, for example a terminator codon is placed SP-10 Serine codon after.This recombinant clone called after pYASP-10Nter, this dna fragmentation see the generation that decides high-caliber SP-10 polypeptide in being present in E.coli the time.Know also that from other researchs this sequence comprises all aminoacid sequences of SP-10 of the polymorphic form of finding in people's sperm.
B. the Asd that has the terminal SP-10 sequence of C- +The structure of carrier
The SP-10 sequence comprises an ApaLI site at Nucleotide 587 places, in C-end one and terminator codon TAG eclipsed XbaI site.At first use ApaLI enzymolysis SP-10 sequence, strand is outstanding uses the mung bean nuclease enzymic digestion, and the result produces a flush end, first codon coding Pro(dried meat).DNA uses the XbaI enzymolysis then, and archaeal dna polymerase Klenow fragment is mended the life fragment of showing no increases in output, and has the TAG termination codon.PYA3098 is complete with the NcoI enzymolysis, mends flat terminal with Klenow.The dna molecular of these two kinds of modifieds links up by tack, introduces through electroporation to comprise among the χ 6212 of pYA232.The required segmental recombinant plasmid that has correct orientation should be encoded and one be contained 119 amino acid whose products, and wherein 118 propylhomoserin acid are connected on after the initial methionine, and they are encoded by SP-10 fully.The product of this protein fragments and pYASP-10Nter decision has 4 amino acid overlapping.The structure called after pYASP-10cter that this is new.
C.LT-B and SP-10N-end parts fusant make up
As above, LT-B fusion vector pYA3048(comprises a signal sequence, makes product can be transported to pericentral siphon) and pYA3082(lack signal sequence and product stayed in the tenuigenin) can be used for this purpose.Two kinds of vector construction steps are the same.The pYASP-10Nter of Gou Jianing NcoI enzymolysis is then handled with the Klenow fragment and is filled and led up 5 ' protruding terminus as mentioned above.Use the PstI enzymolysis then, with the terminator codon among the pYA3098stop that comprises aforementioned insertion.Carrier pYA3048 and pYA3082 ApaLI enzymolysis, the mung bean nuclease enzymic digestion produces blunt end.Carrier is again with the complete enzymolysis of PstI.Carrier is coupled together with inserting fragment, guide to comprise and be with lacI qAmong the χ 6212 of the pYA232 of gene.The plasmid of structure is called after pYALT-B-SP-10NterPer and pYALT-B-SP-10NterCyt respectively.Studied IPTG induce the back synthetic can with the ability of anti-SP-10 and anti-LT-B antibody response, and the viability of IPTG cell of cultured continuously when existing.Can allow to stablize, the fusion plasmid of high level expression and insertion fragment stable existence is used for following research.If the structure of band signal sequence survive under the IPTG existence condition when cultivating, just ooze confirm the to dash forward existence of LFB/SP-10 fusion rotein in the periplasmic space of shock method and Western engram analysis with cold.Measure fusion rotein then and whether form pentamer at pericentral siphon.The LT-B pentamer is still stablized until 60 ℃ in 0.1%SDS, does not also react with anti-LT-B monomeric igg behind the sds polyacrylamide electrophoresis.The LT-B pentamer in 0.1%SDS to 70 ℃ and on the time depolymerization, produce monomer molecule, just be easy to react with anti-LT-B subunit antiserum(antisera) this moment.Like this, after treatment of different temperature, the test immunogenicity can disclose whether form pentamer.
D. the characteristic research of recombinant clone
As above, four kinds of described recombinant plasmids are comprised band lacI when being present in E.coli χ 6212( qPlasmid pYA232) and S.typhimurium △ cya △ crp △ asd bacterial strain χ 3987(do not comprise lacI qGene) time, carried out comparative studies widely.Checked to add and the growth velocity when not adding IPTG, utilized ELISA and the quantitative Western quantitative level of SP-10 synthetic of having inhaled the seal assay determination.According to these test-results, determined whether make up relevant carrier, energy SP secretion-10Nter and SP-10Cter albumen are to E.coli and S.typhimurium pericentral siphon.Utilize LT-B fusion vector pYA3048, this is easy to accomplish.Because a SacI site is very easily arranged, otch just in time is positioned at after first amino acid coding of the LT-B after the ripe processing, has so just kept complete LT-B signal sequence.Utilize different restriction enzymes point of contact on round pcr and the ready-made plasmid, can make up these recombinant plasmids simply.
Utilize the cold shock of oozing to handle the cell grade separation, measured the position of the gene product of expressing.With the cell cultures quite a while, measure whether there is any genetic instability.(Fig. 1) carries out Computer Analysis to the SP-10 nucleotide sequence, disclosed the existence of forward repetition widely and part inverted repeats.Forward repeats to cause the increase or the minimizing of encoding sequence length, the result who recombinates between child chromosome during as plasmid replication.If there is this genetic instability, just can not use the nontoxic Salmonella bacterial strain of recA, because recA makes the further attenuation of Salmonella.The use of recF mutant has been studied in more definite theory, because the reorganization that the recF sudden change has stoped between plasmid and plasmid is interior.
Measured and cultivated for 50 to 100 generations, whether the existence of DAP, and whether plasmid still keeps.Utilize the pulse-chase method to study proteinic stability.At last, be purifying LT-B/SP-10 fusion rotein, it is measured with combining of GM-1 Sphingolipids,sialo and/or agarose.
3.SP-10 purifying
After the output optimization of LT-B-SP-10 fusion rotein in recombinant avirulent Salmonella, purifying fusion rotein.For fear of polluting LPS, used the △ crp △ asd.S.ty-phimurium LT-2 bacterium χ 4153 that derives, it has galE sudden change, can eliminate LPS core and O antigen and produce.In this bacterial strain, also introduced Tn10 and inserted in the gene of relevant flagellum and first kind cilium, polluted antigen prepd to avoid cilium and flagellar antigen.Utilize the cold the first step of oozing shock as purified fusion protein.This causes relative total protein 15-20 purifying doubly usually.According to quality, use GM-1 affinity column (Tayot etc. 1981) or agarose (Clements and Finkelstein, 1979) to fusion rotein bonded affinity and returnability.Tested the purity of fusion rotein in the purge process, measured total protein, measured fusion rotein with quantitative ELISA with standard method.The fusion rotein of purifying is through the lyophilize prolonged preservation.
4. animal immune
Animal immunity and research have been carried out, described in example 9.
5. band SP-10 sequence synthetic of suitable codon usage frequency to obtain high level expression.
With respect to the codon that the gene of low expression level uses, E.coli and S.typhimurium have tangible preference (IKemura, 1985 to the codon of the gene of high level expression wherein; Gouy and Gautier, 1982).Computer is to the analysis of SP-10 encoding sequence (Fig. 1), disclosed in 265 codons, have on 8 prominent matter and never in E-.coli, use in the gene of high level expression, other 43 in the gene of high level expression frequency of utilization only be that one of percentage is to four.Like this, utilize oligonucleotide synthetic, synthetic 40 to 50 oligomer, it has the suitableeest codon usage frequency, to obtain high level expression in E.coli and S.typhimurium.Utilize pYA3048 or pYA3082(Fig. 9 A and 9B) derivative, merge these oligomer and LT-B then.Carrier can be used MluI and PstLI or ApaI and PstI enzymolysis, clones single chain molecule to utilize the synthetic complementary strand of archaeal dna polymerase Klenou fragment enzymatic.But do before this, be necessary to modify pYA3048(and see Fig. 9 B) in sequence between PstI and the Hind III, insert the translation termination signal at three kinds of reading frames in any.This structure is called carrier pYA3048ter.
For the ease of the epitope location, made up the recombinant clone of a cover band portion overlapping fragments, about 30.SP-10 polypeptide and LT-B sequence merge on it.
Each this clone is determined and can analyze with the ability of the polypeptide of anti-SP-10 antiserum(antisera) reaction.Also used other antiserum(antisera)s, its source is male for vasectomy, or has the male or female of other fertility obstacles, and these may have immune-base.
Also in a recombinant avirulent S.typhimurium vaccine strain, use with the epitope of antibody height reaction, measure them and whether have hyperimmunization originality, excite anti-SP-10 antibody response, or after acrosomal reaction, excite antibody response people's sperm.
6. have an optimum expression, the assembling of stable and immunogenic one synthetic SP-10 encoding sequence
According to above-mentioned result of experiment, a complete S P-10 molecule or its possible big fragment are assembled among the described clone of into top example 10 the 4th joint.
Finish making up after these mutation, including or do not having and carried out the experiment described in the 2nd joint and the 3rd saves among the crowd of fertility obstacle, the SP-10 molecule of purifying is used to carry out secondary immunity, carries out the analysis of anti-SP-10 antibody titers quantitative ELISA.
Example 11
Express the structure of the recombinant avirulent Salmonella of mouse ZP3
As shown in figure 17, made up a LT-B-ZP3 fusant.By annealed synthetic ZP350bp oligomer is linked the pYA3082MluI site, produce plasmid pYA3111(Figure 18).With mouse α ZP3(mouse) serum, inhale the seal method by the bacterium colony immunity and screen about 800 bacterium colonies, choose three positive colonies.These three clones produce a molecular weight be about 14KD in comprise a HpaI site.Then, to comprise the ClaI-PstI fragment that size among the pYA3111 of ZP3 polypeptid coding area is about 0.3kb and introduce ClaI-PstI site among the pYA3048, produce pYA3112(Figure 19), and a LT-B-ZP3 fused protein that has 22 amino acid signal sequences is provided.Though top this two kinds of ZP3 clone expresses the LT-B fused protein of the about 14KD of molecular weight, can with mouse α ZP3 sero-reaction, after fully inducing, the LT-B fusion rotein cell growth of band signal sequence is toxic effect.Therefore, the pYA3112 introduction is contained band IacI qAmong the χ 3987 of the pYA232 plasmid of repressor.In the time of in pYA3112 is present in without any the cell of repressor, the viable cell of appearance has the recombinant chou of LTB-ZP3 fusant; And contain band lacI when being present in qWhen resistance was met among the χ 3987 of pYA232 of thing, fusant gave expression to the product that a size is about 14kd, with LT-B-ZP3 fusant product type among the pYA3111 seemingly.
As shown in figure 17, comprise immundominance B-cell epitope and the eclipsed ZP-3 zone that can cause the epitope of B6AF1 female mice autoimmune oophoritis with it, being included in one section can be by inserting fragment with any ZP-3 of NcoI enzymolysis, replace the oligonucleotide of one section any amino acid needed sequence of decision, and in the dna fragmentation that replaces.For example, the sequence of 6 amino acid asparagus fern acyl Si Sisisi glutamy of coding can be fallen by disappearance.So, this recombinant plasmid can use as mentioned above.
Before with the recombinant plasmid inoculation mouse that selects, to its phenotype and stable the confirmation.The animal immune test is finished by above-mentioned.
According to the non-toxic microbe that foregoing invention provides, be suitable for preparing anti-fertility component.These components help to reduce or prevent user's fertility.The non-toxic microbe that has coding gamete specificity antigen gene also can be used for producing resist the antigenic antibody of expressing in non-toxic microbe, both also polyclonal antibody of mono-clonal.
Figure 911080139_IMG2
Figure 911080139_IMG3
Figure 911080139_IMG5
Figure 911080139_IMG6
Figure 911080139_IMG7
Figure 911080139_IMG11
Figure 911080139_IMG12
Figure 911080139_IMG14
Figure 911080139_IMG16
Figure 911080139_IMG19
Figure 911080139_IMG20
Figure 911080139_IMG21
Figure 911080139_IMG22
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Claims (12)

1, a kind of non-toxic microbe, it comprises the antigenic recombinant expression system of at least a gamete specificity of coding.
2, non-toxic microbe according to claim 1, this non-toxic microbe lacks the chromogene that function is arranged of coding β-aspartate-semialdehyde dehydrogenase (Asd), and then this non-toxic microbe comprises the recombination of encoding function asd polypeptide, this recombination and the antigenic one or more gene linkages of one or more coding gamete specificitys.
3, non-toxic microbe according to claim 1 and 2, this non-toxic microbe comprise the cya gene of a sudden change, and this microorganism can not produce the adenylate cyclase of function in fact as a result.
4, according to the described non-toxic microbe of claim 1,2 or 3, this non-toxic microbe comprises the crp gene of a sudden change, and this microorganism can not produce the cAMP receptor protein of function in fact as a result.
5, according to the described non-toxic microbe of claim 1 to 4, this non-toxic microbe belongs to salmonella (Salmonella).
6, non-toxic microbe according to claim 5, this microorganism are Salmonella typhimurium (S.typhimurium).
7, according to the described non-toxic microbe of claim 1 to 4, this microorganism is one intestinal bacteria-Salmonellas (E.coli-Salmonella) hybrid.
8, according to the described non-toxic microbe of claim 1 to 7, its gamete specificity antigen is serum lactic dehydrogenase C, or a kind of polypeptide that contains an epitope of this enzyme.
9, according to the described non-toxic microbe of claim 1 to 7, its gamete specificity antigen is SP-10, or contains the polypeptide of the epitope of SP-10.
10, according to the described non-toxic microbe of claim 1 to 7, its gamete specificity antigen is ZP-3, or contains the polypeptide of ZP-3 epitope.
11, vaccine component, its comprise treatment go up effective dose according to the described non-toxic microbe of claim 1 to 10, connect use with pharmaceutically acceptable mediator.
12, induce the method for anti-fertility state in vertebrates, the indication method comprises the vaccine component according to claim 11 of taking significant quantity to the indication animal.
CN91108013A 1991-11-18 1991-12-13 Recombinant avirulent salmonella antifertility vaccines Pending CN1072454A (en)

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