WO2018007726A1

WO2018007726A1 - Kit for detecting a protein biomarker in a biological sample and amplifying the signal

Info

Publication number: WO2018007726A1
Application number: PCT/FR2017/051764
Authority: WO
Inventors: Ghislain Yannick GANGWE NANA
Original assignee: Gangwe Nana Ghislain Yannick
Priority date: 2016-07-05
Filing date: 2017-06-30
Publication date: 2018-01-11
Also published as: FR3053700B1; FR3053700A1

Abstract

The present invention concerns a method for detecting a biomarker in a biological sample, a plasmid, a bacteria and kits for doing same. The present invention is characterised by a modified lactic acid bacteria, said lactic acid bacteria having at least one histidine kinase receptor and at least one sequence coding for a reporter gene, said histidine kinase receptor being chimeric and the extracellular domain of said receptor comprising a biomarker binding domain, said chimeric receptor being activated by the natural ligand and/or the biomarker of same, and the cytoplasmic domain of said receptor activating a specific signalling cascade of said receptor capable of activating the expression of the reporter gene. In one embodiment, the system according to the invention further comprises an amplifying molecule allowing the biomarker detection signal to be amplified. Advantageously, the biomarker is a protein.

Description

KIT FOR DETECTING A PROTEIN BIOMARKER IN A

BIOLOGICAL SAMPLE AND SIGNAL AMPLIFICATION

Field of the invention

The present invention relates to the field of in vitro diagnostic kits for the detection of biomarkers of pathologies in a biological sample. More specifically, the present invention is in the field of protein detection techniques, including low concentration, quantification of proteins in a biological sample and amplification of the detected signal.

Context of the invention

In general, the earlier a pathology is detected in a patient, the greater the likelihood of healing, since management will be better and more effective.

Pathologies such as cancer or autoimmune diseases require a rapid diagnosis to consider a background treatment. In general, a physical examination allows the practitioner to suspect a cancer that can then be confirmed by imaging and diagnosed by biopsy following the study of genes, proteins and other specific factors. More generally, in recent years, early diagnoses have been developed to take care of patients as soon as possible and thus increase the chances of recovery.

The establishment of a diagnosis is based in particular on molecular biology techniques based either on the antibody / antigen interaction such as the ELISA method (Enzyme-linked Immunosorbent Assay), the western blot or the immunohistochemistry, or on the study. genetics by PCR (Polymerase Chain Reaction) techniques, quantitative PCR, sequencing techniques or cytogenetics (karyotype, in situ hybridization). Among these methods currently used, karyotyping, in situ hybridization techniques, PCR, sequencing allow in certain cases to recognize the pathology in the case of characteristic chromosomal anomaly, but do not however highlight additional genetic abnormalities and post-translational modifications of proteins. PCR and sequencing, although highly sensitive by signal amplification, are limited to gene analysis. This limits their use in diagnosis because indeed, there is no absolute correlation between the mRNA expression levels and the expression of the corresponding protein (Gygi et al., 1999). Moreover, it is impossible to deduce the functional situation of a pure protein from its level of expression. Therefore, technologies that will facilitate direct protein analysis are needed.

State of the art There are state of the art methods of protein analysis:

Among these methods, immunoassays are still the preferred platform for most protein studies, especially clinical diagnoses and drug development, where specificity is critical.

The specificity of the Western blot can be very high. However, Western blots are limited to the detection of the denatured protein, since all the proteins in the sample are denatured before the electrophoresis step of separating the proteins according to their molecular weight. In addition, the protein transfer step on the membrane is technically complex, requiring many steps and a relatively large volume of sample, and therefore are not easily automated. This transfer step is relatively long and expensive. The Western Blot is a good qualitative technique but is very limited from a quantitative point of view. Only a comparison with an internal control can be done but it is purely indicative of a trend. ELISA tests are technically less difficult than Western blots, and can be adapted to higher throughput with plate processing and automated detection systems, but like the Western blot, they offer only single-blot results protein per test. Unlike the Western blot, an ELISA can be used to detect intact natural proteins. ELISA tests can be quantitative when run with a standard curve of a known protein, and well characterized antibodies. However, these tests are not very accurate and also require many steps and a relatively large sample volume. The limit of detection of a protein in a biological sample by ELISA is of the order of pg / ml to several ng / ml.

Protein chips offer advantages of higher throughput, multiplex analysis, low reagent consumption, sensitivity and lower sample quantity requirement compared to ELISA or Western Blot tests. However, the use of protein chips is generally not feasible routinely by a hospital practitioner. Protein chip tests are more suitable and used for basic research and drug discovery where it is necessary to look at several proteins (> 100), or a "proteome" in a sample. For clinical diagnosis, only a few proteins with controls are sufficient. This approach is limited by the absence of very specific capture reagents. In addition, the current assay is not sensitive enough to measure low abundance proteins. As long as only a limited number of proteins have to be analyzed, protein chips will have difficulty becoming a competitive alternative to conventional methods. (O. Poetz et al., Mechanisms of Age and Development 126 (2005) 161170). As for mass spectrometry methods such as SELDI technology, they are suitable for the rapid detection of differences in the total protein content of different samples. However, they have limitations with respect to high molecular weight proteins or membrane proteins. In addition, the sensitivity of SELDI experiments is much lower compared to sandwich immunoassays (Bandera et al, 2003, Uchida et al, 2002, Zhang et al, 2002). Recently, the hypothesis of exploiting the self-induction systems of bacteria for the purpose of protein detection has been issued by Norris V et al (Norris V et al., The Molecular Chain Reaction, J Mol Microbiol Biotechnol, 2012). . These self-induction systems are nowadays used to produce recombinant proteins in quantity but not for diagnosis.

It is therefore necessary to propose new solutions for reliably, rapidly, safely, highly specific and efficient detection of a protein, even in a very small amount, in a biological sample and amplifying the detected signal.

Solution provided by the invention

The present invention proposes to remedy the disadvantages of the prior art:

by a method for detecting a biomarker in a biological sample by bringing the biological sample into contact with a modified lactic acid bacterium expressing a detection system, optionally a system for amplifying the signal of said biomarker, said biomarker being a protein that can be in very low concentration,

by a plasmid for integrating at least one gene of interest into a bacterium for the expression of the system for detecting and amplifying said biomarker,

and by a modified lactic acid bacterium expressing the detection system, and optionally the system for amplifying the signal of said biomarker.

Thus according to a first aspect, the invention relates to a method for detecting a biomarker in a biological sample comprising the steps of:

contacting the biological sample with a modified lactic acid bacterium, said lactic bacterium having at least one histidine kinase receptor and at least one coding sequence for a reporter gene, said receptor histidine kinase being chimeric and the extracellular domain of said receptor further comprising a binding domain (or portion) of a biomarker, said chimeric receptor being activated by its natural ligand and / or biomarker, and the cytoplasmic portion of said biomarker receptor activating a specific signaling cascade of said receptor capable of activating reporter gene expression

- keeping said bacterium in culture in the presence of the biological sample

measurement of the expression of the reporter gene. It is understood that the modified lactic acid bacterium results from the modification of the nucleic acid content of the lactic acid bacterium by adding a DNA fragment, for example at least one plasmid. It is therefore a genetically modified bacterium.

The term "chimeric" is understood to mean that the receptor comprises a representative portion of the histidine-kinase receptor that can naturally be found in the organism from which it is derived, and a binding domain to a biomarker that the original receptor does not possess. The biomarker can not normally bind to the original histidine kinase receptor. In a preferred embodiment, the chimeric receptor is encoded by a sequence comprising a sequence encoding all or part of the original histidine kinase receptor and a specific binding domain of a biomarker. The original histidine kinase receptor may be of bacterial origin and the biomarker binding domain of mammalian origin, for example human.

In a particular embodiment, the method for detecting a biomarker in a biological sample according to the invention comprises the steps of:

1) bringing the biological sample into contact with a modified lactic acid bacterium so that it expresses a system for detecting and amplifying said biomarker by integrating one or more plasmids comprising a sequence encoding a chimeric NisK receptor; a sequence encoding the intracellular regulatory protein NisR and a coding sequence for a reporter gene under the control of a PnisA and / or PnisF promoter,

2) maintaining said bacteria in culture in the presence of the biological sample, and

3) measuring the expression of the reporter gene. This particular method may further comprise (i) a preliminary step of measuring the expression of the reporter gene as a function of increasing and known amounts of biomarker, and / or (ii) a step of amplifying the detection of the biomarker by activation transcription in said modified lactic acid bacterium of at least one coding sequence for an amplifying molecule.

In one embodiment, the method according to the invention also comprises a preliminary step of measuring the expression of the reporter gene as a function of increasing and known amounts of biomarker. This step makes it possible to establish a standard range from a known biomarker concentration for the determination of the unknown biomarker concentration in the biological sample to be tested.

The biomarker is a protein that may or may not be present in the biological sample depending on the state of the organism from which the sample originated. For example, for the diagnosis of a pathology in humans, the presence of the protein may reveal that the patient has a specific pathology. The method according to the invention makes it possible to detect a protein, even in low concentration, that is to say less than 1 ng / mL, preferably less than 1 μg / mL, more preferably less than 0.1 μg / mL. mL, in the biological sample and amplify the detection signal. The biomarker may, without limitation, be a protein implicated in a human pathology such as a cancer or an autoimmune disease, for example PSA for prostate cancer in humans, IL6, IL1 beta , IL10, IL12, IL15, IL17, IL38 or TNFa for inflammatory and autoimmune diseases in humans. All types of proteins can be detected by the invention. Table 1 lists a number of proteins, without being limiting, which can be detected according to the invention as well as the class of pathology in which they are involved. Similarly, extracellular matrix proteins such as collagen could be detected by the present invention. Protein Type of pathology

HER2 Cancer

mucin 1 (MUC1) Cancer

EGP 40 (EpCAM): Carcinoma marker

Circulating Tumor Cell (CTC)

Mammaglobin: Breast Cancer marker

CTC

TP53 mutated Cancer

TTF-1: CTC marker for lung cancer

EML4-ALK fusion protein Cancer

Kras mutated (G12C) Cancer

Mutated EGFR (L858R) Cancer

Mutated EGFR (T790M) Cancer

tumor-associated antigen L6 Cancer

cytokeratin 8, 18, and / or 19: Cancer

CTC marker

guanylyl cyclase C (GCC): Colorectal Cancer

CTC marker

(p210BCR-ABL) Chronic myeloid leukemia

p185BCR-ABL) Chronic myeloid leukemia

p190BCR-ABL Acute Leukemia

PSCA Prostate Cancer

PSMA: CTC Prostate Cancer marker

HE4 (WFDC2) Cancer

uMAGE-A Skin Cancer

IgA-EMA Celiac Disease

IgA-tTG / IgG-tTG Celiac Disease

anti-cyclic peptides citrullinated Rheumatoid arthritis

(PCC)

anti-keratin antibody (AKA) Rheumatoid arthritis

IgG (MYC) / IgM (MYC) Mycoplasmosis due to M. pneumoniae bacteria

Table 1. Proteins that can be detected, without limitation, according to the system according to the invention

Veterinary applications of the present invention are feasible.

In one embodiment of the invention, the method according to the invention also comprises a step of amplifying the detection of the biomarker by activation of the transcription in the modified lactic acid bacteria of at least one coding sequence for an amplifying molecule. Thus, the method according to the invention comprises the steps of:

contacting the biological sample with a modified lactic bacterium, said lactic bacterium having at least one histidine kinase receptor and at least one coding sequence for a reporter gene, at least one sequence coding for an amplifying molecule, said histidine receptor -kinase being chimeric and the extracellular domain of said receptor further comprising a binding domain of a biomarker, said chimeric receptor being activated by its natural ligand and / or biomarker, and the cytoplasmic portion of said receptor activates a specific signaling cascade of said receptor activating the expression of the reporter gene

- keeping said bacterium in culture in the presence of the biological sample

measurement of the expression of the reporter gene.

The cytoplasmic part can indifferently be called cytoplasmic domain or intracellular domain.

According to a second aspect, the invention relates to a plasmid for transfer into a bacterium comprising:

At least one sequence encoding a histidine kinase receptor,

At least one coding sequence for a reporter gene,

characterized in that said sequence encoding a histidine kinase receptor encodes a chimeric histidine kinase receptor having a binding domain of a biomarker on the extracellular domain of said receptor, said receptor being activated by its natural ligand and / or biomarker and the cytoplasmic portion of said receptor activates a signaling cascade specific for said receptor capable of activating expression of the reporter gene.

In an alternative embodiment, the receptor is a NisK receptor at least a portion of the extracellular domain further comprises a binding domain of a biomarker, said binding domain being at least one of the variable regions of a monoclonal antibody having a high affinity for the biomarker In another variant embodiment, the receptor is a NisK receptor at least a portion of the extracellular domain additionally comprises a binding domain of a biomarker, said binding domain being an extracellular domain for ligand recognition of a receptor whose the ligand is the biomarker. In yet another embodiment, the receptor is a NisK receptor at least a portion of the extracellular domain further comprises a binding domain of a biomarker, said binding domain being the ligand of the biomarker, said biomarker being, in this case, case, a specific receptor of said ligand.

In one embodiment, the plasmid according to the invention also comprises at least one coding sequence for an amplifying molecule, the expression of said amplifying molecule being activated by the signaling cascade activated by the binding of the biomarker to the chimeric receptor. In this embodiment, the plasmid for transfer into a bacterium comprises:

At least one sequence encoding a histidine kinase receptor,

At least one coding sequence for a reporter gene,

At least one sequence coding for an amplifying molecule, characterized in that said sequence encoding a histidine kinase receptor encodes a chimeric histidine kinase receptor whose extracellular domain comprises a binding domain of a biomarker, said chimeric receptor being activated by its natural ligand and / or the biomarker, and in that the cytoplasmic part of said receptor activates a specific signaling cascade of said receptor activating the expression of the reporter gene.

Advantageously, the coding sequence for an amplifying molecule is a coding sequence for the natural ligand of the NisK receptor and / or a coding sequence for a biomarker mimetic peptide synthesized by the modified lactic acid bacterium in the presence of the biomarker.

In a particular embodiment, the plasmid for transfer into a bacterium comprises: i) a sequence coding for a chimeric NisK receptor,

ii) a sequence coding for the intracellular regulatory protein NisR and iii) a sequence coding for a reporter gene under the control of a PnisA and / or PnisF promoter, and is characterized in that at least a portion of the extracellular domain of said NisK chimeric receptor comprises a binding domain of a biomarker, said wherein the chimeric receptor is activated by its natural ligand and / or the biomarker, and in that the cytoplasmic portion of said receptor corresponds to the cytoplasmic domain of a NisK receptor.

In a preferred embodiment, said biomarker binding domain is at least one of the variable regions of a monoclonal antibody having a high affinity for the biomarker; alternatively, said biomarker binding domain is an extracellular ligand recognition domain of a receptor whose ligand is the biomarker.

The plasmid may also further comprise at least one coding sequence for an amplifying molecule, the expression of said amplifying molecule being activated by the signaling cascade activated by the fixing of the biomarker on said chimeric receptor. In a preferred embodiment, the coding sequence for said enhancer molecule is a sequence encoding the natural ligand of the NisK receptor and / or a coding sequence for a biomarker mimetic peptide synthesized by the modified lactic acid bacterium in the presence of the biomarker.

The NICE (Nisin Controlled Gene Expression System) system is the expression system of nisin in lactic acid bacteria. This system has the characteristic of self-regulating. Nisin activates the NisK histidine kinase membrane receptor. Once activated, the NisK receptor phosphorylates the intracellular regulatory protein NisR, the latter activating the PnisA and / or PnisF promoter and therefore the genes under the control of this promoter. This system has been widely used especially in the food industry to produce large quantities of a protein of interest. It is understood that in one embodiment, the plasmid according to the invention comprises the sequence encoding the intracellular regulatory protein NisR, for the chimeric receptor, the coding sequence coding for the reporter gene under the control of a PnisA promoter and / or PnisF and the coding sequence for an amplifying molecule under the control of a PnisA and / or PnisF promoter.

The sequence coding for the biomarker-specific binding zone is inserted at the BAES (sensory histidine kinase in two-component regulatory system) upstream the sequence of histidine kinase A (dimerization / phosphoacceptor zone) according to the scheme of the figure 1 . Advantageously, the biomarker is a protein.

Advantageously, the plasmid is encoded by the sequence SEQ ID 1 and by an insert of a sequence chosen from SEQ ID 2 to 9.

It is understood that the plasmid consists of a basic plasmid pGNSK SEQ ID 1 sequence in which can be inserted at least one of the sequences SEQ ID 2 to 9 coding for at least the chimeric receptor according to the invention on wherein the biomarker of interest can bind and optionally at least one coding sequence for an enhancer molecule and at least one coding sequence for a reporter gene.

The plasmid according to the invention is intended to be inserted into a bacterium, preferably a lactic bacterium, more preferably a bacterium of the Lactococcus lactis type, for the bacterium to express the chimeric receptor, as well as the reporter gene and in one embodiment. that the bacterium further expresses the amplifying molecule under the control of the promoter sensitive to the chimeric receptor signaling cascade. The plasmid according to the invention is intended for the implementation of the method according to the invention for the detection of a biomarker in a biological sample.

In another aspect, the invention provides a modified lactic acid bacterium for detecting a biomarker in a biological sample comprising:

At least one histidine kinase receptor At least one coding sequence for a reporter gene

characterized in that said histidine kinase receptor is chimeric and said receptor further comprises a binding domain of a biomarker located on the extracellular domain of the receptor, said receptor being activated by its natural ligand and / or the biomarker,

and in that the cytoplasmic portion of said receptor activates a signaling cascade activating expression of the reporter gene.

In a first embodiment, the chimeric histidine kinase receptor is a NisK receptor at least a portion of the extracellular domain further comprises a binding domain of a biomarker, said binding domain being at least one of the variable regions of a monoclonal antibody having a high affinity for the biomarker.

In a second embodiment, the chimeric histidine kinase receptor is the NisK receptor at least a portion of the extracellular domain further comprises a binding domain of a biomarker, said binding domain being an extracellular ligand recognition domain. a receptor whose ligand is the biomarker.

In yet another embodiment, the chimeric histidine kinase receptor is a NisK receptor at least a portion of the extracellular domain further comprises a binding domain of a biomarker, said binding domain being the ligand of the biomarker, said biomarker being, in this case, a specific receptor of said ligand.

Advantageously, the reporter gene is placed in front of a promoter activatable by NisR. It is understood that the reporter gene is chosen from GFP, GusA, luciferase, lacZ, CobA or any other reporter gene known to those skilled in the art and that the latter is placed in front of a promoter activatable by NisR, NisR being activated by the NisK-c receptor after fixation of the biomarker on NisK-c. The reporter gene according to the invention is preferably under the control of a PnisA or PnisF promoter. When the chimeric receptor according to the invention is activated by the biomarker, NisR is phosphorylated and phosphorylated NisR will activate the transcription of the reporter gene under the control of PnisA or PnisF. Preferably, the reporter gene is chosen from the luminescent reporter genes. The luminescent reporter genes for the practice of the present invention are luciferase.

In one embodiment, the modified lactic acid bacterium further comprises at least one coding sequence for an amplifying molecule, the expression of said amplifying molecule being activated by the binding of the biomarker to the chimeric receptor. The sequence coding for an amplifying molecule according to the invention is preferably under the control of a PnisA or PnisF promoter. In this embodiment, the modified lactic acid bacterium for detecting a biomarker in a biological sample comprises:

At least one histidine kinase receptor

At least one coding sequence for a reporter gene

At least one coding sequence for an amplifying molecule

characterized in that said histidine kinase receptor is chimeric and said receptor further comprises a binding domain of a biomarker on the extracellular domain of said receptor, said receptor being activated by its natural ligand and / or the biomarker,

Advantageously, the amplifying molecule is the native ligand of the histidine kinase receptor and / or the biomarker and / or a biomarker sequence recognizable by the histidine kinase receptor. The histidine kinase receptor capable of recognizing a sequence of the biomarker, the biomarker and / or the native ligand is the Nisk receptor and / or the chimeric NisK-c receptor.

Advantageously, the modified lactic bacterium is a Gram + bacterium selected from the genera Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Tetragenococcus, Leuconostoc, Oenococcus, Bifidobacterium, preferably Lactococcus. Even more preferably, the lactic acid bacterium is a Lactococcus lactis.

The lactic acid bacteria of which Lactococcus lactis are non-invasive and non-pathogenic bacteria widely used in the manufacture of dairy products, including cheese. Lactococcus lactis is a well-characterized bacterium, as is Escherichia coli or Bacillus subtilis.

In another variant, the modified bacterium is a Gram + bacterium such as Bacillus sp. Thus it is understood that the modified lactic acid bacterium according to the invention comprises at least one plasmid according to the invention as described above. The bacterium may include one of the following combinations:

At least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, at least one copy of a plasmid comprising the genetic material for the expression of the reporter gene

At least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, at least one copy of a plasmid comprising the genetic material for the expression of the reporter gene, at least one copy of a plasmid comprising the genetic material for the expression of the amplifying molecule

At least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, the bacterium already comprising a reporter gene controlled by the chimeric receptor-activated signaling pathway;

At least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, the bacterium already comprising a reporter gene controlled by the chimeric receptor-activated signaling pathway and an amplifying molecule of which synthesis is activated by biomarker detection At least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, at least one copy of a plasmid comprising the genetic material for the expression of the amplifying molecule, the bacterium containing already a reporter gene controlled by the chimeric receptor-activated signaling pathway

at least one copy of a plasmid comprising the genetic material for the expression of the chimeric receptor according to the invention, for the expression of the reporter gene, for the expression of the amplifying molecule.

It is understood that in one embodiment, the lactic acid bacterium expresses the system for detecting and amplifying the biomarker by integrating at least one copy of a plasmid comprising the sequence coding for the intracellular regulatory protein NisR, the chimeric receptor, the coding sequence for the reporter gene under control of a PnisA and / or PnisF promoter and the coding sequence for an amplifying molecule under the control of a PnisA and / or PnisF promoter.

According to one variant embodiment, the lactic bacterium expresses the biomarker detection and amplification system by integrating at least one copy of a plasmid containing a coding sequence for the reporter gene and at least one copy of a plasmid containing a sequence encoding the chimeric receptor and a sequence coding for the amplifying molecule. The plasmid encoding the reporter gene can be co-transfected with the plasmid encoding the chimeric receptor and the amplifying molecule or be present constitutively in the bacterial strain used or in the same plasmid as the gene encoding the chimeric receptor and the amplifying molecule. . In this variant embodiment, the reporter gene is under the control of a promoter sensitive to the chimeric receptor signaling cascade, either under the control of a PnisA and / or PnisF promoter.

The modified lactic acid bacterium allows the implementation of the method according to the invention. According to a third aspect, the present invention relates to kits for detecting a biomarker in a biological sample.

According to a first variant, the kit for the detection of a biomarker in a biological sample comprises at least one modified lactic acid bacterium according to the invention

the culture medium of said bacterium

- a standard.

The bacteria present in the kit may be in the form of a pellet, suspended in a culture medium, taken in a culture gel, frozen or lyophilized. Preferably, the bacteria are freeze-dried and to be re-cultured after rehydration.

We understand that the standard is a biomarker or the amplifying molecule.

According to a second variant, the kit for the detection of a biomarker in a biological sample comprises:

at least one plasmid according to the invention

- a standard.

It is understood that the kits of the invention may comprise transfection reagents, lactic acid bacteria, lactic acid bacteria culture medium, 96-well culture plates, microfluidic systems. The present invention has the advantages of reducing the number of steps to be carried out by the experimenter, of drastically reducing the time to obtain the results, and of having a greater limit of detection, in particular thanks to his control system. amplification of the signal by a chain reaction. In addition, the present invention is transposable to the detection of any type of protein, including when present in very small amounts in a biological sample. By amplifying the signal, even a small amount of protein can be detectable by amplification of the signal. The technology of the present invention also has the advantage of being affordable and potentially more profitable than state-of-the-art techniques. By the possibility of having receptors instead of an antibody, this system offers the possibility of analyzing proteins for which antibodies do not currently exist.

The present invention can be used in monoplex, to detect a single protein, or in a multiplex, to detect several proteins simultaneously.

The applications of the present invention can be:

- diagnostic applications intended for humans or animals, including early diagnosis,

- the therapeutic orientation and the stratification of pathologies for a better therapeutic orientation

the monitoring of pathologies by quantification of the biomarker at different times by the object of the invention

the evaluation of the response of a patient to a therapeutic solution.

The pathologies concerned by the present invention may in particular be oncological, inflammatory or autoimmune pathologies.

The present invention will be better understood in the light of nonlimiting examples of embodiment.

Brief description of the figures:

Figure 1a shows a NisK-c design scheme by genetic modification of native NisK reception.

Figure 1b shows a schematic of plasmid pGNSK.

Figure 1c shows an example of an insertion scheme of the NisK-c receptor in the plasmid pGNSK.

FIG. 1 d represents an example of an insertion scheme of the amplifying molecule and then of Nisk-c in the plasmid pGNSK. In FIGS. 1c and 1d: the number of base pairs corresponds to that of the PSA specific system according to the invention.

FIG. 2 represents the verification of the authenticity of the plasmid by BamHI enzymatic digestion in the colonies transformed with at least one plasmid according to the invention.

FIG. 3 represents the measurement of fluorescence in the absence and in the presence of PSA in bacteria incorporating the plasmid according to the specific invention of the detection of PSA.

FIG. 4 represents the measurement of fluorescence in the absence and with increasing doses of IL6 in bacteria incorporating the plasmid according to the specific invention of the detection of NL6.

FIG. 5 represents a test for determining the limit of detection of NL6 by fluorescence measurement of bacteria incorporating the plasmid according to the invention, specific for the detection of NL6 in the absence and in the presence of increasing doses of IL6.

Definitions:

BAES: NisK receptor histidine kinase transduction signal

Biomarker: The biomarker is a protein that may or may not be present in the biological sample depending on the state of the organism from which the sample originated. The biomarker is a measurable biological characteristic related to a normal or pathological process.

Ery: resistance to Erythromycin

HATPase_c: histidine ATPases of the NICE system. The intracellular domain (HATPase_c) of NisK or Nisk-c phosphorylates the intracellular regulatory protein NisR which then activates the PnisA promoter and / or the PnisF promoter.

HisKA: histidine kinase A (dimerization / phosphoacceptor) of the NisK receptor IL6: Interleukin 6: cytokine involved in the acute phase of inflammation and regulates acute and chronic inflammation.

Nisin: Nisin biosynthesis sensor protein: receptor histidine kinase membrane of nisin. The intracellular domain (HATPase_c) of NisK phosphorylates the intracellular regulatory protein NisR which then activates the PnisA promoter and / or the PnisF promoter.

NisK-c: chimeric histidine kinase receptor according to the invention

NisR: Intracellular regulatory protein of the system, phosphorylated by NisK or NisK-c. Once phosphorylated activates the PnisA promoter and the PnisF promoter. pGNSK: basic plasmid according to the invention in which an insert containing at least one NisK-c receptor according to the invention can be inserted

PnisA and PnisF: promoters activated by NisR phosphorylated by NisK or Nisk-c and triggering downstream gene synthesis

PSA: prostate-specific antigen: the prostate specific antigen is a protein made exclusively by the prostate serving to liquefy semen. This protein is strongly expressed in the case of benign hypertrophy or prostate cancer in humans. repD: replication gene D, repE: replication gene E, repF: replication gene F, repG: replication gene G chimeric receptor: receptor according to the invention being a histidine kinase receptor not recognizing the biomarker naturally and comprising a field of biomarker recognition. The chimeric receptor is also capable of activating a signaling pathway triggering the synthesis of the reporter gene and optionally an amplifying molecule. TNFcx: tumor necrosis factor: cytokine involved in systemic inflammation and in the acute phase of inflammation. Deregulation of this cytokine is observed in many autoimmune diseases. EXAMPLES

Method for obtaining the synthetic genes encoding chimeric NisK-c receptors:

Different receptors chimeric NISK-c according to the invention were constructed (GeneArt ^® Gene Synthesis, Thermofisher). These receptors are encoded by synthetic genes. They comprise: the cytoplasmic domain of the native NisK receptors responsible for the initiation of signal transduction which leads to the activation of NisR a recognition domain of the biomarker of interest that may correspond to:

o either to the variable regions of the heavy chain and the light chain of a monoclonal antibody specifically recognizing the biomarker of interest

o the extracellular part of a transmembrane receptor specifically recognizing the biomarker

o is the binding domain of the biomarker on signaling molecules of the cytokine or hormone type

o the ligand of a membrane receptor (when the membrane receptor is the biomarker),

the PnisF promoter

restriction sites Ndel, Ncol

The Ncol site at the end of PnisF later allows transcriptional fusion with a gene that may be a reporter gene.

These nonlimiting examples of synthetic genes are summarized in Table 1 and are encoded by the sequences SEQ ID 2 to 7. Gene name Biomarker Description N ° of synthetic to detect sequences

Nap7 PSA Nisk-anti-protein 7 (NisK-c model 7) SEQ ID 2 specific to PSA, PnisF with sites

Ndel, Ncol restriction

NapT7 TNFa Nisk-anti-protein 7 (NisK-c model 7) SEQ ID 3 specific for TNFa, PnisF with site

Ndel, Ncol restriction

Nap6 PSA Nisk-anti-protein 6 (NisK-c model 6) SEQ ID 4 specific to PSA, PnisF with site

Ndel, Ncol restriction

NapT6 TNFa Nisk-anti-protein 6 (NisK-c model 6) SEQ ID 5 specific for TNFa, PnisF with site

Ndel, Ncol restriction

OPnapIR IL6 Nisk-anti-protein 7 (NisK-c model 7) SEQ ID 6 specific NL6, PnisF with site

restriction Ndel, Ncol

OPnapl7 IL6 Nisk-anti-protein 7 (NisK-c model 7) SEQ ID 7 specific to NL6, PnisF, PnisA, with

restriction site Ndel, Ncol, Fsel,

plus the sequence coding for NL6

with the signal sequence of Usp45

Table 1. Examples of synthetic genes encoding a chimeric receptor

NISK-c

The synthetic genes Nap7, NapT7, Nap6, NapT6 of Table 1 combine the native NisK cytoplasmic domain with the variable regions of the heavy and light chain of a monoclonal antibody specific for the biomarker. The OPnapIR synthetic gene combines the cytoplasmic domain of native NisK with the extracellular portion of a transmembrane receptor specifically recognizing the biomarker.

The synthetic gene OPnapl7 furthermore comprises an enhancer consisting of a sequence coding for NL6 plus a sequence for an extracellular secretion signal Usp45 (see section "synthetic genes coding for the signal amplifiers" below). Synthetic genes encoding signal amplifiers:

In these exemplary embodiments of the invention, the system comprises a signal amplifier induced by the binding of the biomarker on NisK-c capable of specifically recognizing the biomarker. This enhancer is encoded by a synthetic gene having a sequence coding for an extracellular secretion signal, Usp45 in the case of the bacterium Lactococcus lactis. Table 2 lists, without limitation, the synthetic genes of the amplifiers.

Table 2. Examples of Synthetic Genes Encoding Amplifiers

The OPnapl7 gene combines a NL6-specific NisK-c coding sequence and an NL6 and Usp45 enhancer coding. The sequence for this synthetic gene is identified in Tables 1 and 2 by the same sequence number, SEQ ID 7, since it is the same sequence.

Generation of the Plasmid Containing the Gene Encoding the NisK-c Chimeric Receptor and an Amplifier A plasmid pGNSK was constructed by insertion of a synthetic fragment comprising an erytromicin resistance gene (Em), an origin of replication pAMbl (rep E, rep G, rep F, rep D), a gene coding for NisR, a PnisF promoter, the NdeI and Fsel restriction sites) in the vector pBluescript II SK via the Smal cloning site (Genecust ®, Luxembourg). This plasmid is capable of replicating in gram + and gram- bacteria. This plasmid pGNSK is the basic plasmid of the invention in which an insert containing at least one coding sequence for a NisK-c receptor according to the invention can be inserted. The plasmid pGNSK has the sequence number SEQ ID1.

Synthetic genes OPnapl7, OPnapIR, Amnap7 and AmnapT7 are cloned into NdeI-BglII site of plasmid PGNSK ligated (GeneArt ^® Gene Synthesis, Thermofisher). The synthetic genes (NAP7 NAP6, NAPT7, NAPT6) are cloned via the Ndel-Fsel site of the plasmid PGNSK or pGNAmnaP or pGNAmnaPT by the method of seamless cloning using the assembly kit geneart ^® Seamless plus Cloning and Assembly Kit in accordance with the recommendations of the merchant (Thermofisher). The fluorescent reporter gene cobA is cloned via the Ncol-Fsel site of plasmid PGNAPI.

Table 3 summarizes the plasmids used or obtained according to the invention.

Table 3. Plasmids used and obtained according to the invention Propagation and verification of plasmids

The resulting plasmids (Table 3) are propagated in E.coli (in accordance with the manufacturer's recommendations (geneart ^® Seamless plus Cloning & Assembly Kit, Thermofisher)): 3 μΙ of the mixture of the assembly reaction (ligation) is added to a flask of chemically competent E. coli bacteria (One Shot DH10B ™ T1 R SA Thermofisher). In parallel, 3 μl of the pGNSK control DNA is added to a separate flask of chemically competent E. coli bacteria (One Shot ™ DH10B T1 R SA, Thermofisher). The transformation mixture is incubated on ice for 30 minutes then transferred to a water bath at 37 ° C for exactly 10 minutes and then again on ice for 2 minutes. 250 μl of SOC medium is added at room temperature to the transformation mixture and then stirred horizontally (200 rpm) at 37 ° C. for 1 hour. After incubation, 5-150 μί of each transformation are distributed on a preheated selective plate (LB with 100 g / ml of ampicillin). The plates are incubated overnight at 37 ° C. The next day, individual colonies are collected. Their plasmid DNA is isolated purified.

Transformant analysis

The colonies are selected and cultured at night in an LB medium containing the antibiotic (100 g / ml of ampicillin). Plasmid DNA is isolated using PureLink® HiPure Plasmid Kits Miniprep (Thermofisher) and their concentration determined by UV spectroscopy. The plasmids are then analyzed by restriction analysis and the results are visualized by agarose gel electrophoresis (E-Gel® EX agarose gel 1%, Invitrogen ™).

The constructs were verified by sequencing (Thermofisher) in accordance with its quality control charter ((GeneArt ^® Gene Synthesis, Thermofisher). The sequence of congruence in the insertion sites was 100%.

Integration of the plasmid into the bacteria

Positive clone plasmids are electroporated in a strain of lactic acid bacterium, Lactococcus lactis MG1363. This strain has the peculiarity of not produce nisin. It does not contain the NisR and NisK regulatory genes. This strain MG1363 is made electrocompetent in SGM17-G medium (medium M17 plus 0.5M saccarose, 2.5% glycine and 0.5% glucose) according to the recommendations of the supplier (Mobitec). Electrocompetent cells are thawed on ice. 50 μl of electrocompetent cells are transferred to a pre-refrigerated electroporation cuvette and then 1 μl of the plasmid (> 100 ng) is added to the electrocompetent cells. Once the DNA is added to the cells, electroporation is done immediately using the Bio-Rad micropulser (2000V, 25 Pf, 200 Ω-Puise (normal reading is 4.5-5 ms)). Immediately after electroporation, 1 ml of G / L-M17 (M17 culture medium (Merck, Millipore) plus 0.5% glucose) + 20 mM MgCl ₂ + 2 mM CaCl ₂ was added to the cuvette at room temperature. The cuvette is then kept for 5 minutes on ice and then incubated for 1-2 hours at 30 ° C. 100 μl and 900 μl of the electroporated cells are plated at 30 ° C. on M17 + agarose (1.5%) containing 0.5% (weight / volume) of glucose and 0.5 M of sucrose (M17GS) with the antibiotics. selection (5 μg ml Erythromycin for the plasmid with Nisk-c supplemented with 10 μg / ml Chloramphenicol if a second plasmid with the gusA reporter gene pNZ8008 is present).

Verification of the transformation efficiency of the positive clones in Llactis Six colonies were chosen and cultured overnight in 5 ml of M17G medium (10 μg ml of Erythromycin for the plasmid with Nisk-c supplemented with 10 μg ml Chloramphenicol if a second plasmid with the gusA reporter gene pNZ8008 is present). The next day, 5 ml of the fullgrown culture are diluted in 25 ml of M17G medium and incubated for 3 hours at 30 ° C. The tube is then centrifuged for 10 min at 6000 g. The pellet is taken up in 10 ml of the R3 suspension solution (PureLink® HiPure plasmidKits Midiprep, Thermofisher) plus 40 mg / ml of lysozyme in the reaction tube. Resuspension is incubated for 30 minutes at 37 ° C. The plasmid DNA is then isolated (PureLink® HiPure plasmidKits Miniprep (Thermofisher)). The isolated plasmids are analyzed after restriction by BamHI enzymatic digestion according to the indications of the kit (Anza ™ Restriction Enzyme Cloning System, Invitrogen ™) and revelation on agarose gel. The first colony corresponds to the size marker (E-Gel® 1 Kb Plus DNA Ladder, Invitrogen ™) and each column corresponds to an isolated colony. The empty plasmid (pGNSK) served as a control.

Figure 2 shows that 67% of the colonies after transformation with the plasmid pGNAP6 gave positive clones, 75% of the colonies after transformation with the plasmid pGNAP7 gave positive clones. 100% of the colonies after transformation with the plasmid pGNT6 and pGN06 gave positive clones.

The indicator strains (positive clones) are then grown overnight in M17G (M17 medium plus 0.5% glucose) supplemented with the selection antibiotic (10 g / ml Erythromycin for the plasmid with Nisk-c supplemented with 10 g / ml Chloramphenicol if second plasmid present (pNZ8008)). Then 1 ml of glycerol (60%) is added to 3 ml of culture and the cells are stored at -80 ° C for long-term storage.

Validation of the specific induction of the reporter gene by stimulation of the bacteria with the biomarker

This step aims to: a) demonstrate that the reporter gene is induced by the biomarker of interest. For this, the bacteria are brought into contact with the biomarker of interest and the induction of the expression of the reporter gene under the control of the PnisA or PnisF promoter which are normally naturally induced in the presence of nisin is followed.

b) Demonstrate that there is no induction when the biomarker of interest is absent from the culture medium in order to show the specificity of the system, c) Demonstrate that this detection makes it possible to quantify the biomarker

The protein of interest (human PSA (merkmillipore) or recombinant human IL6 or recombinant human TNFα (Thermofisher) according to the bacterial strain) is prepared in 0.1% Tween 80 dissolved in distilled water acidified to pH 2.5 with HCl to prevent adsorption of the protein onto the surfaces of the polypropylene tube so that the concentration of the interest in the culture medium ranges from 1 to 1000 μg / ml. - Validation of the ability of a NisK-c to recognize its ligand and activate NisR and consequently the promoters inducible by the latter

o reporter gene under the control of the PnisA promoter: functional analysis by detection of the j3-glucuronidase activity

Plasmids were introduced into Lactococcus lactis MG1363 by electroporation as previously described harboring plasmid pNZ8008 (NIZO, Mobitec), a pNZ273 derivative which contains a transcriptional fusion of the GusA reporter gene encoding β-glucuronidase to the PnisA promoter. The resulting transformed bacteria are designated U-NGN. The indicator strains were plated on plates of GM17-X-Gluc (M17 [Merck, Germany] agar media supplemented with 1% glucose and 0.5 mM 5-bromo-4-chloro-3-indolylglucuronide ( X-Gluc, Thermofisher) plus chloramphenicol (5 μg / ml) and erythromycin (3 μg / ml) in the presence or absence of 1 μg / ml of the protein of interest The cells are cultured at 30 ° C. overnight. Transformants that showed resistance to chloramphenicol (5 mg / ml) and erythromycin (3 mg / ml) were easily obtained.

Bacteria containing the chimeric NisK-c receptor plasmid according to the invention gave rise to white colonies on GM17-X-Gluc plates (M17 [Merck, Germany] supplemented with 1% glucose and 0.5 mM 5-bromo-4-chloro-3-indolyl-glucuronide (X-Gluc, Thermofisher) when the protein of interest was not added to the plates or when the plasmid pGNSK did not contain the modified gene (example of PSA In contrast, blue colonies were obtained when these transformants were plated on GM17-X Gluc plates in which the protein of interest was included (eg PSA or NL6).

Therefore, β-glucuronidase activity (reporter gene) remained below detection limits under non-induction conditions (no blue colony formed in lack of specific biomarker) and raised to high levels (blue colony formation) by addition of the protein of interest in the culture medium according to the type of Nisk-C receptor present in the plasmid. The results are summarized in Table 4.

Table 4. Expression of the GusA reporter gene in bacteria comprising at least one plasmid for the detection of IL6 according to the invention PnisF-associated reporter gene for the detection of PSA FIG.

The cells used in this test are L. lactis MG1363 bacteria integrating the PGN06 plasmid comprising the PSA-specific NAP6 gene and the cobA reporter gene fused to PnisF (indicator bacterium U-GNO6). Cells then stored at -80 ° C are diluted 1: 25 in M17 medium supplemented with glucose (0.5%), and supplemented with erythromycin (10 μg / ml). Cells are cultured at 30 ° C without aeration to an optical absorbance of 0.16 to 600 nm. The bacterial suspensions are then divided into 50 μΙ portions in a 96-well microtiter plate. Next, 150 μl of PSA (1 ng / ml) prepared in 0.1% Tween 80 (pH 2.5) or 150 μl of 0.1% Tween 80 (pH 2.5) without PSA (0 ng) as a control are added to each well. The cells are incubated for 3 h at 30 ° C. and then the fluorescence is read. The fluorescence is expressed in% EMI as determined with a plate reader (Flx-Xenius XM Thermocontrolled Multimode Microplate Reader (SAFAS)). The experiment was carried out with four different bacterial colonies with a repetition of three parallel wells for each colony.

The results obtained are shown in FIG. 3. The indicator cells without the protein of interest, in other words the controls (0 ng / ml of PSA), had a fluorescence of 20% EMI. In the presence of 1 ng / ml of PSA in the culture medium, the fluorescence increased significantly to 31.40% EMI (the standard deviation is the result of three experiments performed in parallel). The experiment made it possible to show that the presence of PSA in the medium induced the production of the cobA reporter gene placed under the control of the PnisF promoter by the system with the PSA specific Nisk-c receptor.

Transformants harboring the plasmids according to the invention comprising the chimeric receptors according to the invention express genes under the control of nisin-inducible promoters such as PnisA, PnisF in Lactococcus lactis MG1363.

These results demonstrate that the system according to the invention is functional. - Dose-response relationships in L. lactis MG1363 harboring the plasmid pGNAPI _C obA (U-GNIC): FIG. 4

Cells are cultured in the evening and kept in culture on the night. These cells growing (optical absorbance of about 0.7 at 600 nm) were diluted 1: 25 in culture medium M17 supplemented with glucose (0.5%), 10 ^"3 M aminolevulinic acid and supplemented with erythromycin (10 μg / ml) The cells are cultured at 30 ° C. without aeration for 1 h 30 to 2 h until an optical absorbance at 600 nm measured with a spectrophotometer of between 0.2 and 0.3.

The bacterial suspensions are then divided into 50 μΙ portions in a 96-well microtiter plate. Various solutions of IL6 at increasing concentrations (0, 15, 30, 60 and 125 μg / ml) are prepared in 0.1% Tween 80 (pH 2.5). 150 μΙ of these solutions are distributed in dedicated wells. Then the 96-well plate is sealed and placed in the Flx-Xenius XM (SAFAS) thermocontrolled multimode microplate reader at 30 ° C. The measurement of fluorescence as well as the Bacterial growth is performed every hour for 7 hours (beginning of the stationary phase). The excitation wavelength is 357 nm and the emission wavelength is 605 nm. The fluorescence is expressed in% EMI. The results are shown in Figure 4A (n = 3). The background is the well containing cells with a solution of 0 ng / mL IL6. The background fluorescence values were subtracted from the measured fluorescence values for the other IL6 concentrations to obtain relative fluorescence values (RUF) (measured value divided by the background noise value). The lowest IL6 concentration for which a signal was detected received a RUF value of 1 to which the other detection measurements at higher concentrations were reported.

A dose-response fluorescence using the indicator bacterium U-GNIC was obtained from the first hour (linear correlation coefficient R ² = 94) after induction with different concentrations of IL6 (Figure 4A). Concentrations of 15 to 125 μg / ml have been reliably and quantitatively and stably detected even 7 hours after induction (Fig. 4B) .The optimal fluorescence levels as a function of time induction have been determined (Figure 4C). The activity of the PnisF promoter was measured at several time points after the addition of IL6 solution at 125 μg / ml. It has been observed that the promoter activity rapidly increases exponentially after induction with the IL6 solution, the maximum level being reached 4h to 7h after induction (Fig. 4C).

The system according to the invention therefore makes it possible, in addition to the detection, to quantify biomarkers using the concentration standards and this in a short time. . Test of the detection limit of the Nisck-c system: example of L. lactis MG1363 harboring the plasmid pGNAPI _∞ bA (U-GNIC)

The IL6 bioassay used in this study consisted of the following steps: For this test, IL6 concentrations ranging from 0 to 1000 μg / ml IL6 (final concentration of the assay mixture) are added (Fig. a sample of U-cells GNIC thawed and diluted 1: 100 in M17G (more than 10 ^"3 M aminolevulinic acid), followed by overnight incubation at 30 ° C the next day 150 μΙ of the supernatant are removed, followed by freezing at - 20 ° C (30min) then thaw and fluorescence measurement Fluorescence is expressed in% EMI as determined with a plate reader (Flx-Xenius XM Thermocontrolled Multimode Microplate Reader (SAFAS)) All IL6 assays were replicated twice, each replicate contained three parallel wells for each IL6 concentration, and from the repeated experiments and well replication, the lowest IL6 concentrations (minimum detection limits) were determined as values. relative fluorescence units (RUs) that were greater than the fluorescence conditions without adding the biomarker.

In general, the fluorescence signal remains below the detection limits under non-induction conditions and is increased to high levels by the addition of small amounts (> 1 μg) of IL6 in the culture medium (FIG. ), while presenting a dose-linear relationship. The cultures having been left overnight, the signal reaches saturation and given the amplification of the signal by the system, the low concentrations of induction end up giving a signal almost equivalent to that obtained by the higher concentrations. Because the amplification of the signal is dose-dependent until the saturation of the signal, the system can therefore in principle allow the detection of even single molecules.

This new analysis has significantly detected lower amounts of IL6. The major advantage of this sensitivity is that the samples can be diluted widely prior to the test, thus allowing the use of very small amounts of sample. These results demonstrate that the biomarker-inducible system can be implemented in lactic acid bacteria such as Lactococcus lactis and that stimulation with the biomarker specifically induces the reporter gene, and this in a dose-dependent manner in the case of a system without an amplifying molecule or to detect a protein even in a very small quantity with a system having an amplifying molecule.

Claims

1 - Method for detecting a biomarker in a biological sample comprising the steps of:

bringing the biological sample into contact with a modified lactic bacterium, said lactic bacterium expressing at least one histidine kinase receptor and comprising at least one coding sequence for a reporter gene, said histidine kinase receptor being chimeric and the extracellular domain of said receptor gene; chimeric receptor further comprising a binding domain of a biomarker, said receptor being activated by its natural ligand and / or biomarker, and the cytoplasmic portion of said receptor activating a signaling cascade specific for said receptor capable of activating gene expression protractor

- keeping said bacterium in culture in the presence of the biological sample

measurement of the expression of the reporter gene. 2 - The method of claim 1 characterized in that it further comprises a prior step of measuring the expression of the reporter gene according to increasing amounts and known biomarker.

3 - Process according to claim 1 or 2 characterized in that it further comprises a step of amplifying the detection of the biomarker by activation of the transcription in the modified lactic acid bacterium of at least one coding sequence for an amplifying molecule.

4 - Plasmid for transfer into a bacterium comprising:

At least one sequence encoding a histidine kinase receptor,

At least one coding sequence for a reporter gene, characterized in that said sequence encoding a histidine kinase receptor encodes a chimeric histidine kinase receptor comprising a binding domain of an extracellular domain biomarker of said receptor, said receptor being activated by its natural ligand and / or the biomarker, and in that the domain cytoplasmic said chimeric receptor activates a specific signaling cascade of said receptor capable of activating the expression of the reporter gene.

5 - Plasmid according to claim 4 characterized in that the receptor is a NisK receptor whose extracellular domain further comprises a binding domain of a biomarker, said binding domain being at least one of the variable regions of a monoclonal antibody having a strong affinity for the biomarker.

6 - Plasmid according to claim 4 characterized in that the receptor is a NisK receptor whose extracellular domain further comprises a binding domain of a biomarker, said binding domain being an extracellular domain for ligand recognition of a receptor whose the ligand is the biomarker.

7 - Plasmid according to any one of claims 4 to 6 characterized in that it further comprises at least one coding sequence for an amplifying molecule, the expression of said amplifying molecule being activated by the signaling cascade activated by fixation biomarker on the chimeric receptor. 8 - Plasmid according to claim 7, characterized in that the sequence coding for an amplifying molecule is a coding sequence for the natural ligand of the NisK receptor and / or a coding sequence for a mimetic peptide of the biomarker synthesized by the modified lactic acid bacterium in the presence of the biomarker. 9 - Plasmid according to any one of claims 4 to 8 characterized in that it is encoded by the sequence SEQ ID 1 and an insert of a sequence selected from SEQ ID 2 to 9.

10 - Modified lactic acid bacterium for the detection of a biomarker in a biological sample comprising: at least one histidine kinase receptor

At least one coding sequence for a reporter gene characterized in that said histidine kinase receptor is chimeric and the extracellular domain of said receptor further comprises a binding domain of a biomarker, said chimeric receptor being activated by its natural ligand and / or biomarker, and that the cytoplasmic of said receptor activates a signaling cascade capable of activating the expression of the reporter gene.

1 1 - modified lactic acid bacterium according to claim 10, characterized in that the chimeric histidine kinase receptor is a NisK receptor whose extracellular domain additionally comprises a binding domain of a biomarker, said binding domain being at least one of the regions variables of a monoclonal antibody with high affinity for the biomarker.

12 - modified lactic acid bacterium according to claim 10 characterized in that the chimeric histidine kinase receptor is the NisK receptor whose extracellular domain further comprises a binding domain of a biomarker, said binding domain being the extracellular domain of recognition of the ligand of a receptor whose ligand is the biomarker.

13 - modified lactic acid bacteria according to any one of claims 10 to 12 characterized in that the reporter gene is placed under the control of a promoter activatable by NisR.

14 - modified lactic acid bacteria according to any one of claims 10 to 13 characterized in that it further comprises at least one coding sequence for an amplifying molecule, the expression of said amplifying molecule being activated by fixing the biomarker on the chimeric receptor.

15 - Modified lactic acid bacterium according to claim 14 characterized in that the amplifying molecule is the native ligand of the histidine kinase receptor and / or the biomarker and / or a biomarker sequence recognizable by the histidine kinase receptor.

16 - modified lactic acid bacterium comprising the plasmid according to claims 4 to 9 17 - Kit for the detection of a biomarker in a biological sample comprising

at least one modified lactic acid bacterium according to claims 10 to 16

the culture medium of said bacterium

- a standard (amplifying molecule or biomarker)

18 - Kit for the detection of a biomarker in a biological sample comprising:

at least one plasmid according to claims 4 to 9

- a standard