Screening and identification of H-2 d-restricted T cell epitopes in fusion and attachment glycoproteins of Nipah virus by ELISPOT / 中华微生物学和免疫学杂志

Objective:To screen and identify H-2 d-restricted T cell epitopes in fusion (F) and attachment (G) glycoproteins of Nipah virus (NiV) in mice. Methods:The complete peptides (single peptide contains 15 amino acids, and 10 amino acids were repeated in the front and back peptides) derived from F and G antigens were mixed into peptide libraries. BALB/c mice were immunized with DNA vaccines expressing NiV F and G proteins alone and in combination. The full sequence peptide libraries of F and G antigens were mixed into peptide pools by matrix design, and spleen cells of immunized mice were collected and analyzed by IFN-γ ELISPOT assay to detect the dominant H-2 d-restricted epitope peptides. Results:Twelve dominant H-2 d-restricted peptides were screened from the F protein-specific peptide library and the 56th peptide produced the strongest reaction. Four dominant peptides were screened from the G protein-specific peptide library and the 72nd peptide produced the strongest reaction. Conclusions:In this study, 12 F antigen-specific and 4 G antigen-specific H-2 d restricted dominant T cell epitopes of NiV were screened and identified by IFN-γ ELISPOT, which could provide reference for immunological analysis of NiV and vaccine research.

Theoretical Verification of Dynamic Lumbar Interspinous Process Stabilization Device with Memory Alloys / 中国医疗器械杂志

To find an effective method of minimally invasive treatment combining prevention and assistance in the middle zone of conservative treatment and fusion surgery for lumbar degenerative diseases, through the clinical effect observation and the advantage and disadvantage comparison of several commonly used lumbar interspinous dynamic stabilization systems, by analyzing the physiological structure, biomechanics and relevant data of lumbar interspinous processes, based on fully understanding of memory alloy materials, a new dynamic lumbar interspinous fixation device with a memory alloy material has been independently designed and researched, which can not only reconstruct the normal biomechanical characteristics of the lumbar spine and satisfy the normal activities of the human spine, but also avoid damage to the original structure and reduce the incidence of postoperative complications. In addition, the device can be used to optimize the current therapeutic methods. According to our research, the dynamic lumbar interspinous process stabilization device with a memory alloy has theoretically achieved satisfactory results, which can be used to overcome the shortcomings of the existing technology and is superior to the current several dynamic lumbar interspinous process stabilization systems.

Effect of polydatin on the proliferation and apoptosis of THP-1 cells and the mechanism / 中国当代儿科杂志

OBJECTIVES@#To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism.@*METHODS@#After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting.@*RESULTS@#After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 μmol/L. After treatment with 1 800 μmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05).@*CONCLUSIONS@#Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

Normalization and parallel comparison of the detection results of antibodies against SARS-CoV-2 / 中华微生物学和免疫学杂志

Objective:To improve the consistency of test results through reducing inter-laboratory variation in SARS-CoV-2 antibody detection with WHO SARS-CoV-2 antibody candidate international standard (IS, sample G) and antibody reference panel (samples E, F, H, I, J).Methods:Ten WHO samples (A-J) including the candidate IS and reference panel were evaluated using different methods, such as microneutralization tests based on live SARS-CoV-2, pseudovirus neutralization assay and commercial ELISA kits. The test results were compared using statistical analysis.Results:Using IS (sample G) as a reference, the relative concentrations of other samples could be determined with less variation. ELISA and pseudovirus neutralization assay had consistent results with those obtained with the microneutralization test based on SARS-COV-2 strain HB02. Weakly positive samples could be detected only by a certain kit.Conclusions:The availability of an IS for antibodies would facilitate the standardization of SARS-CoV-2 antibody detection methods. The reference panel fitted all the assays based on the SARS-CoV-2 prototype Wuhan strain. Pseudovirus neutralization assay and ELISA could be used as alternatives to live SARS-CoV-2-based neutralization test to some extent.

Construction of a novel bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 and evaluation of its immunogenicity in mice / 中华微生物学和免疫学杂志

Objective:To construct a bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 and to evaluate its immunogenicity in mice.Methods:The coding sequences for spike 1 (S1) protein of SARS-CoV-2 Beta variant and hemagglutinin (HA) of influenza A virus Cambodia (H3N2) strain were codon-optimized and synthesized. The two coding genes were ligated by the self-cleaving 2A peptide using over-lapping PCR to construct S1-2A-HA fragment, which was inserted into pVRC vector to construct the bivalent DNA vaccine, named as pVRC-S1-2A-HA. Indirect immunofluorescence assay (IFA) and Western blot were performed to detect the expression of S1 and HA proteins. BALB/c mice were immunized with pVRC-S1-2A-HA by intramuscular injection and electroporation. The humoral immune responses induced in mice were detected by indirect ELISA, pseudovirus neutralization assay and hemagglutination inhibition assay. Cellular immune responses were detected by IFN-γ ELISPOT, intracellular cytokine staining (ICS) and cytometric bead array (CBA).Results:The bivalent DNA vaccine pVRC-S1-2A-HA could express S1 and HA proteins in vitro. Specific cellular immune responses against S1 protein and specific IgG antibody against HA protein were significantly induced in mice with single-dose immunization. The antigen-specific immunity was significantly enhanced after booster immunization. The geometric mean titer (GMT) of specific IgG antibody increased to 3 251 for S1 protein and 45 407 for HA protein after two-dose immunization. Moreover, the S1-specific T cells increased to 1 238 SFC/10 6 cells. ICS results indicated that the booster vaccination induced CD4 + T and CD8 + T cells to produce IL-2, IFN-γ and TNF-α in mice. The secretion of various cytokines including IL-2, IL- 4, IL-6, IL-10 and IFN-γ in mouse splenocytes was induced after single-dose immunization. Conclusions:A bivalent DNA vaccine against SARS-CoV-2 and influenza A virus H3N2 was constructed and could induce S1- and HA-specific humoral and cellular immune responses in mice, suggesting the great potential of it for further development and application.

Co-inheritance of G6PD deficiency and 211 G to a variation of UGT1A1 in neonates with hyperbilirubinemia in eastern Guangdong.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, which may manifest as neonatal hyperbilirubinemia, is the most prevalent erythrocytic enzyme-related disease in the world. OBJECTIVE: To investigate the association between neonatal hyperbilirubinemia and co-inheritance of G6PD deficiency and 211 G to A variation of UGT1A1 in Chaozhou city of eastern Guangdong province, the effects of G6PD deficiency and UGT1A1 gene variant on the bilirubin level were determined in neonates with hyperbilirubinemia. METHOD: The activity of G6PD was assayed by an auto-bioanalyzer. PCR and flow-through hybridization were used to detect 14 common G6PD mutations in G6PD deficient neonates. 211 G to A variation of UGT1A1 was determined by PCR and sequencing. The data of neonatal bilirubin was collected and analyzed retrospectively. RESULTS: Seventy four cases of the 882 hyperbilirubinemia neonates were G6PD deficiency (8.39%) while 12 cases of the 585 non-hyperbilirubinemia neonates (control group) were G6PD deficiency (2.05%). The rate of G6PD deficiency in the hyperbilirubinemia group was higher than that of the control group. Moreover, the peak bilirubinin of the G6PD-deficient group of hyperbilirubinemia neonates was 334.43 ± 79.27 µmol/L, higher than that of the normal G6PD group of hyperbilirubinemia neonates (300.30 ± 68.62 µmol/L). The most common genotypes of G6PD deficiency were c.1376G > T and c.1388G > A, and the peak bilirubin of neonates with these two variants were 312.60 ± 71.81 µmol/L and 367.88 ± 75.79 µmol/L, respectively. The bilirubin level of c.1388G > A was significantly higher than that of c.1376G > T. Among the 74 hyperbilirubinemia neonates with G6PD deficiency, 6 cases were 211 G to A homozygous mutation (bilirubin levels 369.55 ± 84.51 µmol/L), 27 cases were 211 G to A heterozygous mutation (bilirubin levels 341.50 ± 63.21 µmol/L), and 41 cases were wild genotypes (bilirubin levels 324.63 ± 57.52 µmol/L). CONCLUSION: The rate of G6PD deficiency in hyperbilirubinemia neonates was significantly higher than that of the non-hyperbilirubinemia neonates in Chaozhou. For the hyperbilirubinemia group, neonates with G6PD deficiency had a higher bilirubin level compared to those with normal G6PD. For hyperbilirubinemia neonates with G6PD deficiency, there was a declining trend of bilirubin levels among 211 G to A homozygous mutation, heterozygous mutation, and wild genotype, but there was no significance statistically among the three groups.

Human serum albumin variants in China: a molecular epidemiological investigation and literature review.

BACKGROUND: Bisalbuminemia is a hereditary and/or acquired abnormality characterized by a double albumin (ALB) band on serum protein electrophoresis. However, there have been no epidemiological investigations of ALB variants in Chinese populations. METHODS: This retrospective study examined 71,963 unrelated subjects from five provinces in southern China. ALB variants were screened by cellulose acetate electrophoresis at pH 8.6 and ALB mutations were confirmed by polymerase chain reaction-DNA sequencing. RESULTS: The average incidence of inherited bisalbuminemia in the southern Chinese population was 0.0264% (19/71,963). Thirteen cases showed slow and six showed fast genetic variants on cellulose acetate electrophoresis. Four kinds of ALB variants were identified: proalbumin Lille (p.Arg23His), ALB Castel di Sangro (p.Lys560Glu), ALB Fukuoka-1 (p.Asp587Asn), and a novel ALB Wuxi (p.Lys562Glu). The gene frequency of ALB variants in the Wuxi region (0.126%, 13/10,297) was significantly higher than in other regions in southern China, and 90.9% (10/11) of cases of proalbumin Lille were also found in the Wuxi region. CONCLUSIONS: This study provides the first report of the detailed prevalence and molecular characterization of ALB variants in southern China. Compared with other areas of China, Wuxi had a different pattern of ALB variants and a high prevalence of proalbumin Lille.

[Phenotype and Genotype Analysis in Two Pedigrees with Hereditary Coagulation Factor â ª Deficiency].

OBJECTIVE: To analyze the phenotype and genotype in two pedigrees with hereditary coagulation factor Ⅺ (FⅪ) deficiency, and investigate the molecular mechanisms of FⅪ deficiency. METHODS: Two patients with hereditary coagulation FⅪ deficiency were admitted to Chaozhou Central Hospital in Nov 2014 and Jan 2018. The prothrombin time (PT), activated partial thromboplastin time (APTT), FⅪ activity (FⅪ∶C) and FⅪ antigen (FⅪ∶Ag) were tested for phenotypic diagnosis. All the exons and exon-intron boundaries of FⅪ gene of proband were analyzed by PCR and sequencing. The family members were tested for the mutant site of proband. Then the mRNA of FⅪ in the proband was analyzed with RT-PCR. RESULTS: The proband-1 was a 7-year-old boy, PT was 10.7 s and APTT was 97.4 s (reference range: 9-12.8 s; 24-40 s), FⅪ∶C (0.6%) and FⅪ∶Ag<1% (reference range: 65%-150%; 72.1%-122.3%). The proband-2 was a 30-year-old female, and showed the PT (11.7 s), APTT (71.3 s), FⅪ∶C (0.7%) and FⅪ∶Ag<1%. FⅧ∶C, FⅨ∶C and FⅫ∶C of two proband were within the normal range. DNA sequencing showed that the proband-1 had a combined mutation of c.326-1G>A and c.1107C>A (p.Tyr351X) in exon 10. His grandmother, mother and brother had a heterozygous splicing mutation of c.326-1G>A, his grandmother and father had a homozygous mutation of c.1107C>A. FXI mRNA was undetected in the proband-1. The proband-2 had a homozygous mutation of c.841C>T (p.Gln263X) in exon 8, and this mutation was also found in her father, mother, daughter and son. CONCLUSION: The c.326-1G>A, c.1107C>A(p.Tyr351X) and c.841C>T (p.Gln263X) might be the molecular pathogenesis for two probands with hereditary coagulation factor Ⅺ deficiency.

Gene variants in four pedigrees with hereditary coagulation factor XI deficiency and one novel mutation identification.

: Coagulation factor XI (FXI) deficiency is a bleeding disorder with unpredictable severity. Patients with this condition usually suffer bleeding manifestations after trauma or surgery and are poorly correlated with plasma FXI activity (FXI:C). In the current study, we examined and identified the phenotype and genotype in four unrelated probands and their 32 relatives with hereditary FXI deficiency. The probands with severely reduced FXI:C but bleeding symptoms were only found in two probands. Mutation analysis showed that all the probands were FXI homozygous mutation or compound heterozygous mutation. Five mutations were identified including three nonsense mutations c.841C>T (p.Gln263X), c.1107C>A (p.Tyr351X) and c.1033A>T (p.Lys327X), respectively, one frameshift mutation c.1325delT (p.Leu424CysfsX8), and one splicing mutation c.326-1G>A. c.1033A>T (p. Lys327X), a novel mutation which lead to a premature stop codon at amino acid position 327, it may have an influence on protein characteristics and cause the corresponding disease.

Liposomal Irinotecan for Treatment of Colorectal Cancer in a Preclinical Model.

Colorectal cancer (CRC) is the most frequently diagnosed cancer and leading cause of cancer-related deaths worldwide. Because of the use of first-line CRC treatments, such as irinotecan (IRI), is hindered by dose-limiting side effects, improved drug delivery systems may have major clinical benefits for CRC treatment. In this study, we generate and characterize liposomal irinotecan (Lipo-IRI), a lipid-based nanoparticle, which shows excellent bioavailability and pharmacokinetics. Additionally, this formulation allows IRI to be maintained in active form and prolongs its half-life in circulation compared to IRI in solution. Compared with IRI statistically, the level of prostaglandin E2 (PGE2) in colonic tissue decreases, and Bifidobacterium spp. (beneficial intestinal microbiota) content increases in the Lipo-IRI-treated group. Moreover, no damage is observed by the hematoxylin and eosin staining of the normal tissue samples from the Lipo-IRI-treated group. In a xenograft mouse model, CRC tumors shrink markedly following Lipo-IRI treatment, and mice receiving a targeted combination of Lipo-IRI and liposomal doxorubicin (Lipo-Dox) extend their survival rate significantly. Overall, our results demonstrate that this formulation of Lipo-IRI shows a great potential for the treatment of colorectal cancer.

Design and Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine Hybrids as Potent Anticancer Agents that Inhibit Angiogenesis and Induce DNA Interstrand Cross-links.

Hybrid molecules are composed of two pharmacophores with different biological activities. Here, we conjugated phthalazine moieties (antiangiogenetic pharmacophore) and bis(hydroxymethyl)pyrrole moieties (DNA cross-linking agent) to form a series of bis(hydroxymethyl)pyrrolo[2,1- a]phthalazine hybrids. These conjugates were cytotoxic to a variety of cancer cell lines by inducing DNA damage, arresting cell cycle progression at the G2/M phase, triggering apoptosis, and inhibiting vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells. Among them, compound 29d encapsulated in a liposomal formulation (e.g., 29dL) significantly suppressed the growth of small-cell lung cancer cell (H526) xenografts in mice. Based on immunohistochemical staining, the tumor xenografts in mice treated with 29dL showed time-dependent decreases in the intensity of CD31, a marker of blood vessels, whereas the intensity of γ-H2AX, a marker of DNA damage, increased. The present data revealed that the conjugation of antiangiogenic and DNA-damaging agents can generate potential hybrid agents for cancer treatment.

Serological assay to detect human antibodies against monkey poxvirus / 中华实验和临床病毒学杂志

Objective@#To establish a method for detection of human antibodies against monkeypox virus.@*Mothds@#The enzyme linked immunosorbent assay (ELISA) plates were coasted with two monkeypox virus peptides from B21R protein, to establish an indirect ELISA for detecting monkeypox virus IgG antibody. The healthy individuals serum samples, monkeypox virus infected patient serum samples and other virus infected patient sera samples were applied to evaluate specificity of the peptides antigen. The reaction conditions were optimized.@*Results@#Synthesized two peptides from monkeypox virus BR21R protein did not cross react obviously with healthy person serum and other virus infected serum. It was shown that the reaction condition was best with sera dilution at 1∶50 when two combined peptides were coated at 100 ng /well, and second-antibody was diluted at 1∶20 000. At this condition the cut off value of IgG antibody in serum samples for ELISA were A450 reading of 0.393. The detected results of two serum samples collected from the monkeypox patient in Sierra Leone were strongly positive, the titers of IgG antibody in two sera were both 1∶6 400.@*Conclusions@#The indirect ELISA for detection of monkeypox virus infection was established preliminarily which provided useful tools for epidemiological study and diagnosis.

Combined immunization with human papillomavirus type 18 fusion protein and recombinant vaccinia virus in mice / 中华实验和临床病毒学杂志

Objective@#To investigate the cellular and humoral immune responses induced by combined immunization with the fusion protein of human papillomavirus type 18 (HPV18) and the recombinant vaccinia virus.@*Methods@#Purified HPV18L231-600E7E6 fusion protein, expressed by prokaryotic expression system, were immunized in combination with the recombinant vaccinia virus vaccine expressing HPV18E7E6 fusion protein (rVV18E7E6) by using various prime-boost regiments in C57BL/6 mice. Cellular and humoral immune responses were analyzed by enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and pseudovirus neutralization assay.@*Results@#Higher levels of cellular immune responses were induced in mice primed with the HPV18L231-600E7E6 fusion protein/adjuvant CpG and boosted with the recombinant vaccinia virus rVV18E7E6, than in other immunized mice. Higher binding antibody level was induced, and low level neutralizing antibody against pseudovirus was detected simultaneously.@*Conclusions@#Priming with HPV18L231-600E7E6 fusion protein/CpG and boosting with the recombinant vaccinia virus rVV18E7E6 could induce higher cellular and humoral immune response in immunized mice, which might be taken as vaccine candidate for treatment of HPV18 chronic infection and postoperative adjuvant treatment for cervical cancer.

Preliminary exploration of replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan / 中华实验和临床病毒学杂志

Objective@#To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan.@*Methods@#We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected.@*Results@#We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot.@*Conclusions@#The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Preparation and characteristic analysis of six influenza A (H7N9) pseudovirus derived from different districts of China / 中华实验和临床病毒学杂志

Objective@#To prepare strains of influenza A (H7N9) pseudovirus derived from different districts of China for vaccine efficacy evaluation.@*Methods@#Phylogenetic tree was built based on hemagglutinin (HA) amino acid sequence analyses from 29 influenza A (H7N9) virus strains and 6 influenza A (H7N9) virus strains with HA determinants variation were selected. 293FT cells were co-transfected with plasmid pNL4-3-Luc.R-E-, pVRC-HA and pVRC-NA with codon-optimized hemagglutinin (HA) and neuraminidase (NA) derived from the six influenza A (H7N9) virus strains, respectively. Transmission electron microscopy assay and Western blot analysis were performed to demonstrate morphology and specificity of these particles, luciferase activity assay and hemagglutinin titers detection were used to determine their infectivity and hemagglutinin activity. And finally, pseudovirus-based neutralization assays were evaluated with HA immunized mice serum.@*Results@#Six influenza A (H7N9) peseudovirus particles derived from different districts of China were selected and prepared. All of the particles bearing HA and NA were characterized with classic influenza virus morphology, with TCID50 titer ranged from 104TCID50/50 μl to 105TCID50/50 μl and with hemagglutinin activity ranged from 64 to 512. Neutralization efficacies on influenza A/Shanghai/1/2013(H7N9) HA vaccine serum against 100TCID50 dose of these pseudovirus particles indicated their potential application in the vaccine cross-protective evaluation in future.@*Conclusions@#Six influenza A (H7N9) pseudovirus derived from different districts of China with potential antigenic variation on HA were constructed successfully, established foundation for their further application in vaccine cross-reactive efficacy evaluation.

Generation and characterization of specific monoclonal antibodies against monkeypox virus / 中华实验和临床病毒学杂志

Objective@#To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay.@*Methods@#Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E. coli BL21 to screen antibodies. Synthetic monkeypox virus A2917 ~ 49 polypeptide was used to immune BALB/c mice. Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques. Indirect ELISA was performed to test antibodies specificity and subtype.@*Results@#A29, A27 and 162 proteins were highly expressed in E. coli and detected by Western blot. The three his-tagged proteins were purified using His-Bind affinity chromatography column. The purity of the proteins was all more than 90%. And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins. Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype.@*Conclusions@#Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.

Molecular Epidemiological Characterization and Health Burden of Thalassemias in the Chaoshan Region, People's Republic of China.

Thalassemia is one of the most prevalent inherited disease in southern China. However, there have been only a few epidemiological studies of thalassemia in the Chaoshan region of Guangdong Province, People's Republic of China (PRC). A total of 6231 unrelated subjects in two main geographical cities of the Chaoshan region was analyzed for thalassemia. Seven hundred and thirty-six cases of suspected thalassemia carriers with microcytosis [mean corpuscular volume (MCV) <82.0 fL] were found by complete blood cell (CBC) count, and were tested by reverse dot-blot gene chip to reveal a total of 331 mutant chromosomes, including 278 α-thalassemia (α-thal) alleles and 53 ß-thalassemia (ß-thal) alleles. The most common α-thal mutations were the Southeast Asian (- -(SEA)), followed by the -α(3.7) (rightward) and -α(4.2) (leftward) deletions. The two most common ß-thal mutations were HBB: c.316-197C>T and HBB: c.126_129delCTTT, accounting for 69.81% of the ß-thal defects in the studied individuals. In addition, a rare mutation, Cap +1 (A>C) (HBB: c.-50A>C) was described for the first time in the Chaoshan region. Our results gave a heterozygote frequency of 5.31% for common α- and ß-thal in the Chaoshan region, and also indicated a higher prevalence of thalassemia with a heterozygote frequency of 6.29% in Chaozhou, followed by Shantou (3.37%). This study provided a detailed prevalence and molecular characterization of thalassemia in the Chaoshan region, and will be valuable for developing a strategy for prevention of thalassemia and reducing excessive health care costs in this area.

[From random mutagenesis to precise genome editing: the development and evolution of genome editing techniques in Drosophila].

Drosophila melanogaster, an important model organism for studying life science, has contributed more to the research of genetics, developmental biology and biomedicine with the development of genome editing techniques. Drosophila genome-editing techniques have evolved from random mutagenesis to precise genome editing and from simple mutant construction to diverse genome editing methods since the 20th century. Chemical mutagenesis, using Ethyl methanesulfonate (EMS), is an important technique to study gene function in forward genetics, however, the precise knockout of Drosophila genes could not be achieved. The gene targeting technology, based on homologous recombination, has accomplished the precise editing of Drosophila genome for the first time, but with low efficiency. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated precise genome editing is simple, fast and highly efficient compared with the gene targeting technology in Drosophila. In this review, we focus on Drosophila gene knockout, and summarize the evolution of genome editing techniques in Drosophila, emphasizing the development and applications of gene targeting, zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9 techniques.

Optimized expression, preparation of human papillomavirus 16 L2E7 fusion protein and its inhibitory effect on tumor growth in mice / 生物工程学报

HPV16 L2E7 is a fusion protein used for therapeutical vaccine targeting HPV virus. To increase its expression in Escherichia coli, we optimized the codon usage of HPV16 l2e7 gene based on its codon usage bias. The optimized gene of HPV16 sl2e7 was cloned into three different vectors: pGEX-5X-1, pQE30, ET41a, and expressed in JM109, JM109 (DE3) and BL21 (DE3) lines separately. A high expression line was selected with pET41a vector in BL21 (DE3) cells. After optimization of the growth condition, including inoculation amount, IPTG concentration, induction time and temperature, the expression level of HPV16 L2E7 was increased from less than 10% to about 28% of total protein. HPV16 L2E7 protein was then purified from 15 L culture by means of SP Sepharose Fast Flow, Q Sepharose Fast Flow and Superdex 200 pg. After renaturing, HPV16 L2E7 protein with ≥ 95% purity was achieved, which was confirmed via SDS-PAGE gel and Western blotting. The combined use of purified HPV16 L2E7 and CpG helper has shown clear inhibition of tumor growth in mice injected with tumor cells, with six out of eight mice shown no sign of tumor. This study lays a solid foundation for a new pipeline of large-scale vaccine production.

High resolution melting analysis: a rapid screening and typing tool for common ß-thalassemia mutation in Chinese population.

ß-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct ß-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify ß-thalassemia. Here, we used an improved HRM method to screen and type 12 common ß-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of ß-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of ß-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.