60 results about "Recombinant baculovirus" patented technology

The invention discloses a method for preparing human metapneumovirus nucleocapsid protein based on an insect-baculovirus system. Comprising the following steps: 1) constructing a recombinant vector with a human metapneumovirus nucleocapsid protein coding sequence: sequentially connecting a sequence for promoting secretory expression, the human metapneumovirus nucleocapsid protein coding sequence and a tag sequence from 5'to 3 'ends in the recombinant vector; 2) converting the recombinant vector with the human metapneumovirus nucleocapsid protein coding sequence constructed in the step 1) into a competent cell containing a baculovirus genome plasmid Bacmid to obtain a recombinant baculovirus genome plasmid; and 3) transfecting insect cells with the recombinant baculovirus genome plasmid obtained in the step 2), culturing and purifying to obtain the human metapneumovirus nucleocapsid protein. The invention provides the preparation method of the human metapneumovirus nucleocapsid protein, and a material support is provided for subsequent further research.

The invention discloses a preparation method of influenza D virus-like particles, and belongs to the field of veterinary biological products. Influenza D virus (IDV) is one of important pathogens of bovine respiratory disease syndrome (BRDC), the disease causes a hundreds of billions of dollars economic loss to the global cattle raising industry every year, however, commercialized vaccines for influenza D do not exist in the market at present. The preparation method comprises the following steps: target gene amplification, construction of recombinant shuttle plasmids, construction of recombinant baculoviruses, rescue of recombinant baculoviruses, and preparation and purification of virus-like particles. The D-type influenza virus-like particles (IDV VLPs) are obtained through the steps. After mice are immunized, the IDV VLPs are found to have high immunogenicity and can be used as a candidate vaccine for preventing the D-type influenza.

The invention discloses a swine fever and porcine parvovirus bivalent subunit vaccine and a preparation method thereof. The vaccine comprises a first recombinant protein encoded by a first gene, a second recombinant protein encoded by a second gene and a pharmaceutically acceptable carrier. The first gene has a sequence as shown in SEQ ID NO: 1 or an increased or reduced sequence thereof. And the second gene has a sequence as shown in SEQ ID NO: 2 or an increased or reduced sequence thereof. An antigen E2 protein of a hog cholera virus (CSFV) and a VP2 protein of a porcine parvovirus (PPV) are taken as double targets, a recombinant SC-E2 protein with a SpyCatcher tag and a recombinant ST-VP2 protein with a SpyTag tag are respectively expressed in insect cells through a recombinant baculovirus vector, double-antigen covalent assembly is realized in vitro, and the constructed bivalent subunit vaccine can be used for simultaneously preventing and controlling two epidemic diseases and has a good application prospect. And the vaccine has the advantages of high safety, strong immunogenicity, high prevention and control efficiency, easiness in large-scale production and the like.

The invention relates to the technical field of biology, and particularly discloses a recombinant vector, a recombinant baculovirus and a preparation method and application of the recombinant vector and the recombinant baculovirus. According to the method for preparing the recombinant vector, pFastBac Dual is adopted as a vector to prepare a recombinant plasmid vector inserted into a VP2 gene of an avian bursal disease virus classic strain, and the recombinant plasmid vector is named as pDual-V1; the method comprises the following steps: preparing a recombinant plasmid vector into which VP2 genes of a classical strain and a variant strain of the avian bursal disease virus are inserted at the same time, and naming the recombinant plasmid vector as pDual-V1-V2; the method comprises the following steps: preparing a recombinant plasmid vector inserted into a group I type 4 fowl adenovirus Fiber-2 gene, and naming the recombinant plasmid vector as pDual-F2; the method comprises the following steps: preparing a recombinant plasmid vector into which a Fiber-2 gene and an avian reovirus sigma C gene sequence are inserted at the same time, wherein the recombinant plasmid vector is named as pDual-F2-sigma C; carrying out double enzyme digestion on the pDual-V1-V2, recovering small fragments, carrying out double enzyme digestion on the pDual-F2-sigma C, recovering large fragments, and then carrying out mixed enzyme ligation. The recombinant baculovirus can express four antigens at the same time, and effective preparation of a multi-combined vaccine is achieved.

The invention discloses a type 1 goose astrovirus virus-like particle and a preparation method and a pharmaceutical composition thereof, and the preparation method comprises the following steps: cloning a gene segment for coding type 1 goose astrovirus ORF2 protein to a baculovirus transfer vector, and constructing to obtain a recombinant plasmid; transforming the recombinant plasmid into a DH10Bac competent cell to obtain a recombinant baculovirus rod grain; transfecting the recombinant baculovirus stem into an insect baculovirus expression system to obtain a recombinant baculovirus; and inoculating the recombinant baculovirus into an insect baculovirus expression system, culturing, collecting a cell culture, and purifying to obtain the purified goose astrovirus type 1 virus-like particles. The GAstV-1 VLPs and the vaccine thereof provided by the invention not only solve the technical problem of research and development of the GAstV-1 vaccine, but also have the advantages of clear structure, high safety, good immunogenicity and exact protective efficacy, and have remarkable creativity and industrial application value.

To provide a method for stably and efficiently producing a protein in secretory production of a target protein using BEVS.SOLUTION: The present invention also provides a method for producing a fusion protein, comprising the steps of expressing the fusion protein in cultured insect cells using a recombinant baculovirus comprising a polynucleotide encoding the fusion protein, and recovering the fusion protein from the culture supernatant, wherein the fusion protein is derived from silkworm H1N1 influenza virus hemagglutinin or silkworm osteonectin.SELECTED DRAWING: None

The invention discloses a recombinant baculovirus transfer vector as well as a preparation method and application thereof. The recombinant baculovirus transfer vector comprises an early promoter and a late promoter which are arranged in sequence, and the downstream of the early promoter and the downstream of the late promoter are independently and operably connected with a porcine circovirus type 2 Cap protein coding frame. According to the recombinant baculovirus transfer vector, the copy number of exogenous genes of a traditional insect vector is greatly increased, the upper limit of the yield of the exogenous genes expressed by the vector is greatly improved, and by carefully designing the arrangement direction and sequence of coding frames of the recombinant baculovirus transfer vector, matching and combination of promoters in the morning and later periods and the like, the yield of the exogenous genes is improved. The high expression of the porcine circovirus PCV2 Cap protein in insect cells is realized.

The invention belongs to the technical field of bioengineering and the field of gene therapy. The invention discloses a baculovirus expression vector for optimizing Rep expression yield, proportion and time. Recombinant adeno-associated viruses with extremely low empty shell rate can be produced in insect cells. A baculovirus early promoter with corresponding strength is selected, and Rep78 / 68 and Rep52 / 40 are expressed at proper strength and proper proportion in the early stage of virus infection (2A self-cleavage polypeptide is used to adjust the promoter strength or virus infection titer). After the Rep expression cassette and AAV2 Cap controlled by a p10 promoter are jointly expressed in insect cells, rAAV particles with the vacant shell rate smaller than 1% can be obtained only by conducting one-step affinity chromatography on a cell lysis solution. Through testing, the strategy can be popularized to AAVs of other serotypes. The low-intensity promoter is used for driving the Rep, so that cell resources are saved, the yield of effective rAAV is remarkably improved due to reduction of the vacant shell rate, meanwhile, the step of removing vacant shell particles can be omitted in the production process due to the ultralow vacant shell rate, and finally the production cost of the rAAV is greatly reduced.

The invention relates to a method for improving PCV2 recombinant baculovirus protein expression, and belongs to the technical field of recombinant virus protein preparation. In order to solve the problem of low expression quantity of the existing PCV2 recombinant virus protein, the invention provides a method for efficiently expressing the PCV2 recombinant virus protein, the method comprises the step of carrying out H5 cell suspension culture for preparation, and we find that when glucose is lower than 5g / L in the culture process, glucose is added to 5-8g / L, glutamine is supplemented to 3-5mM, and beta-mercaptoethanol is added to 0.01-0.1 mM, the expression quantity of the PCV2 recombinant virus protein is increased, and the expression quantity of the PCV2 recombinant virus protein is increased. The expression quantity of the target protein can be obviously increased, and the expression quantity reaches 240 g / L or above. The method is simple in process, condition parameters are easy to control, cell culture is extremely easy to amplify, and the effects of cell culture, virus passage and target protein expression can be achieved without changing liquid in the cell culture period after virus inoculation.

The application discloses a bovine coronavirus virus-like particle, a preparation method and application, and the bovine coronavirus virus-like particle is assembled after co-infection of five kinds of recombinant baculoviruses capable of stably secreting bovine coronavirus E, M, N, S and HE proteins, and contains complete bovine coronavirus E, M, N, S and HE proteins, and BCoV VLPs similar to natural BCoV virus particles in morphology and size are constructed; after bovine coronavirus virus-like particles obtained by the application are used for immunizing mice and calves, high-titer antibody levels can be generated, and cellular immunity of the body can be stimulated.

The invention relates to a method for producing a recombinant baculovirus comprising n exogenous genes in an insect cell, by means of homologous recombination of a replication-deficient baculovirus genome and n transfer vectors, each comprising one of the n exogenous genes, n being an integer at least equal to 2.

The invention belongs to the technical field of biology, and discloses a recombinant turkey herpesvirus, and a tandem epitope expression cassette is inserted between UL45 and UL46 of the recombinant turkey herpesvirus; the tandem epitope expression cassette is used for expressing a plurality of B cell epitopes and a plurality of T cell epitopes. According to the recombinant herpesvirus of turkeys, a tandem epitope is inserted into an HVT (herpesvirus of turkeys) vector, and experiments prove that the recombinant herpesvirus of turkeys can induce higher HI antibody and neutralizing antibody titer, obviously stimulate spleen T lymphocyte response of immunized chicken, and can generate an obvious challenge protection effect in 3 days; the challenge protection effect is obviously superior to that of a combined immunization scheme of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV provided by the applicant; meanwhile, the recombinant turkey herpesvirus is verified to be capable of performing intraembryonic vaccination and generating an obvious immune protection effect. Meanwhile, the invention further provides a preparation method and application of the recombinant turkey herpesvirus.

The invention discloses a method for preparing PRMT2 protein by using a baculovirus expression system and multi-mode chromatography, which comprises the following steps: S1, cloning a base sequence SEQ ID NO.1 of PRMT2 into a plasmid in an artificial gene synthesis mode to form a recombinant expression plasmid; s2, transposing the recombinant expression plasmid obtained in S1 into an escherichia coli competent cell to obtain a recombinant baculovirus, transfecting an insect cell to obtain a baculovirus, and expressing a PRMT2 protein SEQ ID NO.2; and S3, obtaining the purified PRMT2 protein SEQ ID NO.3 through multi-mode chromatography. According to the method disclosed by the invention, the label-free and high-purity PRMT2 protein can be obtained, and a foundation is laid for the biological research of related structures in the future.

The invention belongs to the field of gene engineering, and particularly discloses a recombinant baculovirus vector and an insect cell. The recombinant baculovirus vector comprises a nucleic acid molecule with an exogenous gene expression cassette, the exogenous gene expression cassette is located between a CNE sequence and an NAE sequence of the nucleic acid molecule, and the distance between the exogenous gene expression cassette and the CNE sequence and the distance between the exogenous gene expression cassette and the NAE sequence are both smaller than 5 kb; and the distance between the CNE sequence and the NAE sequence is greater than 1.5 kb. According to the application, the technical scheme that the exogenous gene expression cassette is arranged between the CNE sequence and the NAE sequence of the nucleic acid molecule is adopted, so that the production stability of the exogenous protein or the rAAV vector is better and the yield is higher in continuous passage.

A baculovirus vector and a use thereof in the preparation of a recombinant adeno-associated virus (rAAV) in an insect cell are provided. The baculovirus vector includes an exogenous gene expression cassette and a stable sequence. The stable sequence is located at a site 5 kb or less from the exogenous gene expression cassette, and the stable sequence is a conserved noncoding element (CNE) sequence or a nucleocapsid assembly-essential element (NAE) sequence. When an insect cell is infected with a recombinant baculovirus (rBV) constructed in this way, after multiple continuous passages, production levels of the rBV and the rAAV still remain relatively stable.

A virus-like particle (VLP) includes: an extracellular domain of a hemagglutinin having an amino acid sequence, or the extracellular domain of a spike protein; a matrix protein 1 having an amino acid sequence; and a transmembrane-cytoplasmic domain of the hemagglutinin. A method for preparing the VLP includes: constructing a recombinant plasmid; generating a recombinant bacmid by transformating the recombinant plasmid into a competent cell; generating a recombinant baculovirus by transfecting the recombinant bacmid into a first cell; and harvesting the VLP by transfecting the recombinant baculovirus into a second cell. Also provided is the use of the VLP in preparing a vaccine.

The invention discloses a Bac-to-Bac baculovirus expression system, a recombinant baculosome, a recombinant baculovirus and an application of the recombinant baculovirus. The Bac-to-Bac baculovirus expression system comprises a donor plasmid and a DH10Bac competent bacterium, and is characterized in that the donor plasmid is a recombinant plasmid; the donor plasmid comprises a first temperature-sensitive replicon control element and a heterologous functional gene segment expression cassette; the DH10Bac competent bacteria comprise a baculovirus genome plasmid bacmid and an auxiliary plasmid, and the auxiliary plasmid comprises a second temperature-sensitive replicon control element and a Tn7 transposase expression cassette; the first temperature-sensitive replicon control element and the second temperature-sensitive replicon control element are different. The recombinant bacilli are prepared on the basis of the Bac-to-Bac baculovirus expression system. The recombinant baculovirus is obtained by transfecting the recombinant baculovirus into insect cells and culturing the recombinant baculovirus. The recombinant plasmid prepared by the invention is purer and is not polluted by helper plasmids and donor plasmids.

The invention discloses a micropterus salmoides rhabdovirus gene engineering vaccine preparation method, which comprises: S1, recombinant plasmid construction: carrying out PCR amplification on a micropterus salmoides rhabdovirus G protein gene with a 6 * His tag, cloning the amplified gene between BamH I and Hind III restriction enzyme cutting sites of a pVL1393 vector to obtain a pVL1393-G-His recombinant plasmid, S2, preparing a transfection system, and S3, carrying out purification on the transfection system to obtain the micropterus salmoides rhabdovirus gene engineering vaccine. S3, recombinant baculovirus preparation and identification, S31, cell transfection, S32, P1-generation virus harvesting, and S33, virus identification, the G protein of the micropterus salmoides rhabdovirus (MSRV) is directionally expressed through a genetic engineering technology, a large number of pathogenic viruses do not need to be cultured, the biological safety risk is greatly reduced, meanwhile, the defect that a prokaryotic expression system lacks protein post-translational modification is overcome, and the method is suitable for industrial production. The sf9 insect cell is used as an expression host, so that the recombinant G protein can be correctly folded to form a functional structure domain, and the immunogenicity is remarkably improved.

The invention provides a gene capable of influencing the insecticidal virulence effect of baculovirus and application of the gene, a beet armyworm SeCaspase-4 gene is obtained through screening, a genome bacmid of a recombinant virus carrying the gene is constructed, and a recombinant virus polyhedrosis is successfully obtained through cell transfection. A large amount of expression of the SeCaspase-4 gene can promote cell apoptosis in the baculovirus infection process, and can improve the insecticidal toxicity of the recombinant virus polyhedrosis to lepidoptera insects. Therefore, the recombinant virus is obtained by constructing the recombinant baculovirus bacmid carrying SeCaspase-4 and transfecting insect cells, and the insecticidal toxicity of the recombinant baculovirus on lepidoptera insects can be improved. The invention provides an effective way for improving the insecticidal toxicity of the baculovirus on the lepidoptera insects, and has important significance for efficiently utilizing the baculovirus to carry out biological control on the lepidoptera insects.

The invention discloses a porcine circovirus type 2 Cap protein negative labeled subunit vaccine and a preparation method thereof. On the basis of successful research and development of a PCV2Cap protein subunit vaccine (OKM strain), a recombinant baculovirus is constructed by searching a key amino acid site of a 5H7 monoclonal antibody recognition antigen epitope, a recombinant Cap protein (Cap Y156S) lacking the 5H7 monoclonal antibody recognition antigen epitope is obtained by expression, and electron microscope observation proves that the recombinant Cap protein can form virus-like particles (VLPs). Animal experiments prove that the subunit vaccine can provide good immune protection capacity, vaccine inoculated pigs and naturally infected pigs can be distinguished through a 5H7 blocking ELISA antibody detection method, and the subunit vaccine has important application prospects.

The present invention discloses the use of a recombinant protein in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy. Specifically, it discloses the use of a protein with an amino acid sequence of SEQ ID No.1 in the preparation of a product for diagnosing or assisting in the diagnosis of IgA nephropathy. The present invention uses a recombinant baculovirus containing the gene encoding human glycosyltransferase B3GNT6(43-384) to infect insect cells, obtaining a secreted form of glycosyltransferase B3GNT6(43-384), which can efficiently and specifically recognize the Tn antigen in the hinge region of Gd-IgA1. Based on this, the present invention has developed a novel chemical enzyme method technology for serological diagnosis of IgA nephropathy. This technology can effectively distinguish IgA nephropathy patients, healthy controls, and other nephropathy controls, with high stability, good repeatability, and the characteristics of non-invasive, rapid, specific, and sensitive, having a very broad clinical application prospect and important significance.

To provide a method for easily producing immunogenic pupae for oral administration, and to provide pupae for oral administration produced thereby.SOLUTION: Provided are a method for producing oral pupae, comprising: infecting baculovirus-infectible insect larvae or pupae with a recombinant baculovirus into which DNA encoding an antigen protein has been introduced; and freeze-drying the pupae that have developed from the infected larvae, or the infected pupae.SELECTED DRAWING: None

The invention belongs to the technical field of microorganisms, and particularly discloses a method for preparing self-assembled goose astrovirus vp27 protein-ferritin by using a baculovirus expression system, which comprises the following steps: firstly, constructing a recombinant plasmid pFastBac Dual-VP27 and a recombinant plasmid pFastBac Dual-VP27-ferritin; the preparation method comprises the following steps: respectively transforming a recombinant plasmid pFastBac Dual-VP27 and a recombinant plasmid pFastBac Dual-VP27-Ferritin into a DH10Bac competent cell, so as to obtain a recombinant positive clone bacmid-VP27 and a recombinant positive clone bacmid-VP27-Ferritin, and respectively transforming the recombinant plasmid pFastBac Dual-VP27 and the recombinant plasmid pFastBac Dual-VP27-Ferritin into a DH10Bac competent cell; the method comprises the following steps: respectively transfecting a recombinant positive clone bacmid-VP27 and a recombinant positive clone bacmid-VP27-Ferritin into an insect cell, and carrying out expression of a recombinant baculovirus, so as to obtain a recombinant baculovirus Ac-VP27 and a recombinant baculovirus Ac-VP27-Ferritin; the method comprises the following steps: respectively carrying out subculture on a recombinant baculovirus Ac-VP27 and a recombinant baculovirus Ac-VP27-Ferritin, and carrying out expression of a VP27 protein and a VP27-Ferritin protein; and then separating and purifying the VP27 protein and the VP27-Ferritin protein from the culture supernatant. According to the invention, the VP27 protein and the VP27-Ferritin protein with natural conformations can be conveniently and rapidly obtained, and the VP27 protein and the VP27-Ferritin protein act as candidate vaccines for preventing goose astrovirus infection and antigen substances for detecting antibodies of goose astrovirus.

The invention discloses a fever with thrombocytopenia syndrome virus-like particle, a vaccine and a preparation method of the fever with thrombocytopenia syndrome virus-like particle. Based on SFTSV genome analysis, glycoproteins Gn and Gc and capsid protein NP are selected as core antigens, potential T cell epitopes of the Gn protein are predicted by utilizing bioinformatics, and the chimeric antigen Mosaic-Gn protein is designed. The optimized antigen gene is cloned to a baculovirus transfer vector, and three recombinant baculoviruses are constructed through a homologous recombination technology. The three recombinant viruses co-infect suspended insect cells, and the SFTSV virus-like particles self-assembled in the cells can be harvested after 72 hours. And mixing and emulsifying with an adjuvant to prepare the vaccine after ultra-filtration and ultra-separation concentration purification. The prepared virus-like particle vaccine can induce a body to generate a specific antibody when immunizing a BALB / c mouse, and has the advantages of strong immunogenicity, good safety, high hereditary stability and the like. And the vaccine is verified to have broad spectrum.

The invention discloses preparation of an akabane disease virus N protein monoclonal antibody and a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) detection method, in order to prepare the akabane disease virus nucleocapsid protein monoclonal antibody, an eukaryotic recombinant plasmid pFastBac-AKAV-N is constructed and is converted into a DH10bac competent cell, and a recombinant stem Bacmid-AKAV-N for expressing AKAV N is successfully obtained. After the recombinant baculovirus is transfected to an sf9 insect cell, the recombinant baculovirus capable of stably expressing the AKAV N protein is rescued. A mouse and a rabbit are immunized with the identified and purified recombinant N protein, and a monoclonal antibody and a polyclonal antibody are prepared respectively. The titer of the polyclonal antibody is measured through an ELISA method, and two positive cell strains are obtained through screening and are named as 2G4 and 6C7 respectively. Furthermore, the prepared polyclonal antibody is used as a coating antigen, an HRP-labeled 2G4 monoclonal antibody is used as a detection antibody, and a double-antibody sandwich ELISA detection method for AKAV N protein antigen detection is optimized and established. According to the method, the AKAV N antigen can be specifically recognized, and reliable technical support is provided for clinical antigen monitoring of AKAV. The monoclonal antibody preparation system and the double-antibody sandwich ELISA detection method established by the invention lay an important foundation for pathogen detection and epidemiological investigation of akabane disease.

The invention relates to a method for generating in a single step a library of recombinant baculoviruses for screening for a protein of interest, starting from a pooled-library of poly-nucleotides each encoding a different protein. Insect cells are infected with a pooled-library of baculoviruses at a very low multiplicity of infection (MOI) leading to the situation where the majority of cells are infected by a single baculovirus. The pool of cells is then treated with a viral inhibitor which prevents secreted baculoviruses from penetrating other cells without affecting, either the capacity of the cell to express the target protein, nor intracellular viral replication and accumulation.

A method for preparing protein-based nanoparticle for self-packaging and delivering mRNA includes the following steps. A first donor plasmid, a second donor plasmid and a third donor plasmid are provided. A plasmid transposing step is performed. A recombinant virus preparing step is performed so as to obtain a first recombinant baculovirus, a second recombinant baculovirus and a third recombinant baculovirus. A transducing step is performed, wherein the first recombinant baculovirus, the second recombinant baculovirus and the third recombinant baculovirus are used to infect a producer cell so as to express a nucleocapsid protein, an envelope protein, an engineered envelope protein and a target RNA, and the nucleocapsid protein, the envelope protein, the engineered envelope protein and the target RNA are self-assembled to form a protein-based nanoparticle for self-packaging and delivering mRNA.

Provided are a preparation method and system for a recombinant adeno-associated virus (rAAV) and a recombinant bacmid. The method comprises: first reconstructing a recombinant bacmid containing a recombinant baculovirus genome that produces essential functional elements for an rAAV, at least one of the essential functional elements being inserted into the N-terminal or C-terminal of a locus of an essential gene of the recombinant baculovirus genome; and then transfecting the obtained recombinant bacmid containing the recombinant baculovirus genome that produces the rAAV into a host cell line for culturing to prepare an rAAV. Compared with recombinant baculoviruses obtained by conventional Tn7 recombinant preparations of recombinant bacmid, the recombinant baculovirus obtained by inserting a core element containing Cap, Rep and ITR into two sides of a baculovirus essential gene has a more stable rAAV serial passage production level in a cell and has a higher rAAV yield.

The invention provides a stirring type reactor culture process of a porcine circovirus type 2 recombinant baculovirus, which comprises the following steps: preparing a seed virus of the porcine circovirus type 2 recombinant baculovirus by using an insect cell Sf9 full-suspension culture process, and inoculating an insect cell High Five by using the seed virus prepared by using an Sf9 cell to perform antigen expression of the porcine circovirus type 2 recombinant baculovirus. A full-suspension culture mode is utilized, so that the labor intensity is reduced, the production cost is reduced, and pollution is not easily caused; 2) the risk of porcine virus pollution can be reduced by using insect cells and a baculovirus expression system; and 3) the characteristics (Sf9 and High Five) of different insect cells are simultaneously utilized to respectively carry out seed virus preparation and antigen expression, so that higher virus titer can be obtained, the virus titer is greater than or equal to 7.50 Log (TCID50 / mL), higher recombinant protein expression is obtained, and the protein expression quantity is greater than or equal to 150 mu g / mL.