Abstract:Biological products are a kind of special agents for the prevention, diagnosis, and treatment of diseases. The research and development of biological products come from the combined knowledge of many subjects, such as microbiology, immunology, molecular biology, cytology, genetic engineering and fermentation technology. Biological products industry is the core and the hot spot of the biotechnology industry. In recent years, China has made some advances in biological products research and development. To promote biological products research in China, invited reviews and selected research articles were published in this special issue of “Biological Products”. The reviews and research articles focus on the field of the biological products for the prevention, diagnosis, and treatment of disease.

Abstract:Interferon response is the first line of host defense against virus infection. Recent years have witnessed tremendous progress in understanding of fish innate response to virus infection, especially in fish interferon antiviral response. A line of fish genes involved in interferon antiviral response have been identified and functional studies further reveal that fish possess an IFN antiviral system similar to mammals. However, fish virus-induced interferon genes contain introns similar to mammalian type III interferon genes although they encode proteins similar to type I interferons, which makes it hard to understand the evolution of vertebrate interferon genes directly resulting in a debate on nomenclature of fish interferon genes. Actually, fish display some unique mechanisms underlying interferon antiviral response. This review documents the recent progress on fish interferon response and its molecular mechanism.

Abstract:The technology of genetic engineering has been widely used to express macromolecules such as enzymes. However, it is difficult to detect and purify the micromolecules such as small peptides, because of their instability and degradability. Construction of multi-copy recombinant expression plasmid can be achieved by inserting multiple target genes or expression cassette containing target genes with the same orientation into expression vector. This is effective to increase the expression level of small peptides. In this article we described four methods in order to provide some optional methods and ideas for the expression of active small peptides.

Abstract:Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognising and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various “omics”, especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display. This mini review outlines the genesis, development and application prospect of antibody libraries according to the published reviews and research articles, and offers up to date development and application prospect of antibody library technology.

Abstract:Aptamers are a group of artificial oligonucleotides identified by exponential enrichment system evolution technology (Selective expansion of ligands by exponential enrichment, SELEX). Aptamers have been widely used in basic research, clinical diagnostics, and nano-technology. In this article we will introduce the technology of aptamer and summarize its applications in medical microbiology.

Abstract:As a member of the HSP90 family, heat shock protein (HSP) Gp96 is one of the most abundant proteins in the endoplasmic reticulum (ER), which displayed important molecular chaperones function in cells. Gp96 can stimulate the production of cytokines by activating the antigen presentation cells (such as dendritic cell, et al) in innate immunity. It is capable of eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity. Gp96 is also an ideal adjuvant in many recent researches. Here, we review the progress that addresses the role of biological characteristics, immunogenic mechanism that may be involved in the induction of anti-infection immune response and antitumor immunity, which may guide the new vaccine strategies with the knowledge of Gp96-antigen complexes.

Abstract:Hand-foot-mouth disease (HFMD) is an acute infectious disease caused by various enteroviruses. Recently, large HFMD outbreaks caused by enterovirus type 71 (EV71) have been frequently reported in China, posing great threats on children’s health. There is no specific antiviral therapy for severe HFMD, and patient management mainly depends on supportive and symptomatic treatment. Intravenous immunoglobulin (IVIG) is a pharmaceutical preparation of human IgG that is pooled from thousands of healthy blood donors, and contained neutralization antibodies against various enteroviruses, including EV71. IVIG therapy should be carefully administrated for severe HFMD considering its role on passive immunization against EV71 and immune regulation.

Abstract:Recombinant adeno-associated virus (rAAV)-based vectors that can stably express therapeutic genes in vivo without detectable side-effect have shown great promise for human gene therapy. A major challenge for translation of promising research to clinical development is how to establish clinically compatible purification methods in separating rAAV from potentially pathogenic impurities, especially rAAV vector-related impurities, a class of impurities corresponding to AAV particles that closely resemble bona fide vectors and are difficult to remove. In this review we summarize the assembly process of rAAV vector-related impurities and their characteristics differed with rAAV vectors, and evaluate several current technologies to prevent their formation or separate them from rAAV stocks.

Abstract:Interest in rare diseases research and orphan drugs development has been increased distinctly in recent years. The number of affected people with rare diseases is considerable around the world and the formulation of national and international incentive policies to accelerate orphan drugs development, aiming at offering facilities and necessary conditions for patient access to treatment, gains favorable results. In particular, more measures should be taken to catalyze further progress due to behindhand level in this field in China. Additionally, therapeutic methods of rare diseases were also discussed.

Abstract:Blood products are those biologicals derived from plasma or obtained by recombinant technologies. This overview covers the characteristics and classification of plasma proteins, the current status of products (albumin, immunoglobulins, coagulation factors and microcontent proteins), as well as the likely trends in the near future. Human serum albumin is one of the earliest, safest and most widely used proteins in the pharmaceutical field. The approval and development of high-purity plasma albumin, recombinant human albumin and HSA fusion proteins provide a favorable prospect for the therapeutic protein. Normal immunoglobulin contains antibodies to all the micro-organisms prevalent in the donor population. The IMIG is relatively simple to prepare and use, and the side effects are acceptable; IVIG is used mainly to treat patients with primary immunodeficiency syndromes; SCIG preparations can be used in selecting suitable patients for home therapy and have occurred fewer adverse systemic reactions; specific immunoglobulins contain concentrations of antibody to an individual organism or toxin at a higher titer than normal immunoglobulin and can not be replaced in clinical use. The plasma-derived or recombinant coagulation factors are used to treat the patients with congenital or acquired factor deficiency. The products such as Fibrinogen, FVII, FVIII, von Willebrand complex, FIX/PCC, FXI, FXIII and so on, have been widely used and proved to be effective. The development of recombinant FVIIa is now as a good bypassing product to haemophilia with inhibitors. The Fibrinogen and thrombin play a very important role in surgery hemostasis. Moreover, microcontent proteins including protein C, antithrombin, α1-AT, tPA have been licensed and used in clinical treatment; a number of other small field proteins are under produced research or pre-clinical investment. The ongoing development of new recombinant plasma proteins is providing alternatives for patients, but the distinct position and the potential impact of plasma-derived preparations are unique, furthermore the development of new plasma protein is still a hot spot in global pharmaceutics. Nowadays, a relative difference exists in the development of blood products between our nation and developed countries, so the domestic manufacturers are faced with chances and challenges.

Abstract:Matrix protein 2(M2) is an integral tetrameric membrane protein of influenza A virus, which functions as ion channel. M2 sequence has shown remarkable conservation, so there has been growing interest in it as “universal” vaccine. In order to establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator, M2 gene was obtained by PCR amplification from the plasmid containing the segment 7 of influenza A virus strain A/PR/8/34 firstly. The PCR product was cloned into BamHⅠ/NotⅠ restriction site of pcDNA5/FRT/TO vector, and cotransfected with pOG44 which express Flp recombinase into Flp-In T-REx-293 cell. Integration of pcDNA5/FRT/TO-M2 into the cell genome at the Flp Recombination Target (FRT) site brought the SV40 promoter and the initiation codon in frame with the hygromycin resistance gene. Thus, stable cell lines were selected for hygromycin resistance. The expression of M2 protein from hygromycin-resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay (IFA). 16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator. The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.

Abstract:We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0×106 colonies and the phage library titer was 2.65×1012 TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.

Abstract:To obtain a recombinant human plasminogen (hPLG) with potential anti-platelet aggregation activity, we cloned the cDNA coding Pro544 to Asn791 of hPLG, a kringle-deficit derivative (hPLG-?K). The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp (RGD) motif. The constructed pPICZαA-RGD-HPLG-?K plasmid was expressed in yeast Pichia pastoris GS115, which produced RGD-hPLG-?K about 0.160 g/L broth. After affinity chromatography, the purity of the recombinant protein reached above 90%. Western blotting test confirmed that it retained the immunological reaction capability as human PLG. Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG-?K (P=0.630, n=5). Importantly, after activation by urokinase, RGD-hPLG-?K showed a significantly higher platelet aggregation inhibition rate (Ri) (21.8%±1.57%) than hPLG-?K (3.8%±0.33%) (P=0.000, n=5). These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity, which made a foundation for developing innovative thrombolytic drugs with multifunction.

Abstract:New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.

Abstract:Anthrax is a zoonosis caused by Bacillus anthracis, which seriously affects human health. In recent years, a special phenomenon is found that the metabolic active of a bacterium remains after it is killed. To development of a KBMA (killed but metabolically active) Bacillus anthracis vaccine candidate strain, a plasmid pMAD and a recombinase system Cre-loxP were used to knockout the uvrAB gene of B. anthracis AP422 which lacks both of two plasmids pXO1 and pXO2. The results of PCR and RT-PCR shows that uvrAB genes were deleted from B. anthracis AP422 chromosome successfully. The constructed B. anthracis AP422△uvrAB was inactivated by photochemical treatment (PCT) including an exposure in a long-wave-length ultraviolet (UVA) light and a treatment of 8-Methoxypsoralen (8-MOP), then the metabolic activity were detected by the method of MTS. The results showed that the killed B. anthracis AP422△uvrAB maintained a highly metabolic activity for at least 4 hours, showing a state of KBMA. The KBMA strain of B. anthracis AP422△uvrAB provides the prospective vaccine candidate strain for anthrax.

Abstract:While currently therapeutic vaccines for chronic hepatitis B virus (HBV) infection are actively being developed to complement standard antiviral treatments, their immune activity, especially T cell activity, remains to be further improved. Here, we investigated the role of heat shock proteins HSP70 and gp96 on cellular and humoral immunity, using the main structure antigens of hepatitis core (HBcAg) and surface (HBsAg) as the DNA vaccine. By ELISPOT (enzyme linked immunospot assay), IFN-γ intracellular staining, [3H]-thymidine incorporation and ELISA (enzyme linked immunosorbent assay) analyses, we showed that immunization with HBsAg/HBcAg DNA formulation along with HSP70 or gp96 induced significant increase of T-cell (about 1-6-fold) and antibody (about 20%?60%) immunity against HBsAg and HBcAg. These results may provide bases for designing HSP70- and gp96-based vaccines aimed at eliciting T-cell responses for therapeutic applications.

Abstract:To develop a specific, rapid, and convenient immunochromatography assay (ICA) to detect the rabies antibody in clinical sample from immuned dogs by rabies vaccine. Colloidal gold particles labeled with purified rabies virus (CVS11) were used as the detector reagent. The staphylococcal protein A (SPA) and pured rabbit anti-rabies virus IgG were blotted on the test and control regions of nitrocellulose membrane. Then the strip was assembled with sample pad, absorbing pad, and dorsal shield. The assay samples (261 dog’s serum) were collected from Wildlife Rabies Disease Diagnostic Laboratories of Ministry of Agriculture in China, Institute of Military Veterinary, Academy of Military Medical Sciences and other six provinces, including rabies virus positive and negative serum. The performance of the strip was compared to fluorescent antibody virus neutralization test. The neutralizing antibody titer could be detected above 0.5 IU. The strip did not change of performance when stored at room temperature for 12 months. It may offer reference of neutralizing antibody titer level after dogs immuned rabies vaccine and determin whether the dogs need to be immuned again.

Abstract:In order to detect antibody against swine influenza virus (H1N1), HA1region of hemagglutinin gene in epidemic swine influenza virus (H1N1) strain was amplified and subcloned into prokaryotic expression vector pET30a. Then recombinant HA1 protein was expressed by Escherichia coli BL21. The purified recombinant HA1 protein was obtained after the treatment of denaturing, refolding and affinity chromatography with immobilized nickel chelating NTA (Ni-NTA). An indirect enzyme-linked immunosorbent assay (ELISA) method was established using the purified protein as antigen. Then 785 swine serum samples collected during 2008?2009 were detected by this method, and the positive ratio was 15.54%. There were diversities among provinces (8%?47%). The diagnostic specificity and diagnostic sensitivity of this method arrived at 91% and 95% respectively, using the results of IDEXX ELISA kit as reference.

Abstract:To prepare anti-Brucella abortus serum used for calibrate the agglutination test follwing the national standard, 4 anti-Brucella abortus sera were obtained from 4 cows infected with Brucella abortus naturally. By potency testing, the third serum was selected. Sterility, vaccum degree, residual moisture, uniformity and stability of this standard material were tested and proved to meet the national standard. Referring to the international standard, RBT (Rose-Bengal plate agglutination test), SAT (standard tube agglutination) and CFT (complement fixation test) titers of this standard material were measured to be 1:160 “+”, 1:2 400 “++” and 1:800 “++”, which are identical with the collaborative assay results. International unit of the standard material is 4 000 IU/mL.

Abstract:We selected and characterized isolates of Lactobacillus crispatus (L. crispatus) for potential preventing infections of the female reproductive tract. We cultured vaginal swabs from healthy volunteers on de Man, Rogosa and Sharpe (MRS) agar and identified the isolates at the species level by 16S rRNA sequence and genotyped the isolates of Lactobacillus by PCR amplification of repetitive bacterial DNA elements (rep-PCR). Furthermore, 10 L. crispatus strains were assessed for hydrogen peroxide (H2O2) and acid production. Overall 65 isolates were confirmed to be Lactobacillus by sequence analogy, among them 19 were L. crispatus, 17 were Lactobacillus jensenii and 12 were Lactobacillus fermentum. rep-PCR produced specie and strain-specific genomic fingerprints for the Lactobacillus isolates. The selected 10 L. crispatus isolates produced highly acidic environment after growth in MRS. The isolates T22-3 and T29-5 demonstrated high production of H2O2. This study indicated that there are individual differences with vaginal Lactobacillus colonization, and strain diversity within vaginal L. crispatus isolates, T22-3 and T29-5 might be candidates for restoring urogenital health environment in females.