The detection and clinical feature of HcoV-nL63 in children with acute respiratory tract infection in Lanzhou city / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the prevalence and clinical characterization of HCoV-NL63 (NL63) in children with acute respiratory tract infections (ARTIs) in Lanzhou with other respiratory viruses. The prevalence of HBoV1 in ALRTI was obviously city,China.</p><p><b>METHOD</b>From November 2006 to October 2009,1169 nasopharyngeal aspirates (NPA) were collected from children under 14 years old with ARTIs. Samples were screened for NL63 using a reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Demography and clinical information were recorded.</p><p><b>RESULT</b>NL63 was detected in 35 (2.99%) of the 1169 children. The peak of the positive rate were in August, September 2007, July, August 2008 (23.53%,17.65%, 50%, 33.33% separately). There are no NL63 positive samples was detected in December, 2007 to February 2009. 25 (25/35, 71.43%) were co-infected with other respiratory viruses, and human rhinovirus (HRV) were the most common additional respiratory virus. No significant differences of infective rate of NL63 was found between < or = 3 years age group and > 3 years age group. Bronchiolitis and pneumonia were the most frequent diagnoses in NL63 positive patients and the major symptoms were fever and cough in our study. Between the monoinfection group and the coinfection group of NL63-positive patients, no differences were found in symptoms and clinical diagnoses except symptoms of gastrointestinal.</p><p><b>CONCLUSION</b>HCoV-NL63 is an important pathogen of acute respiratory tract infection in children in Lanzhou city. The peak of HCoV-NL63 infections was in summer. There were annual differences in the prevalence of HCoV-NL63. HCoV-NL63 infections existed a high rate of mixed infection, and mixed infection does not increase the severity of the disease.</p>

Prevalence and clinical characteristics of coronavirus NL63 infection in children hospitalized for acute lower respiratory tract infections in Changsha / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The main objective of this study was to explore the prevalence and clinical characteristics of human coronavirus NL63 infection in hospitalized children with acute lower respiratory tract infection (ALRTI) in Changsha.</p><p><b>METHODS</b>Nasopharyngeal aspirates (NPA) samples were collected from 1185 hospitalized children with ALRTI at the People's Hospital of Hunan province, between September 2008 and October 2010. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to screen for coronavirus NL63, which is a 255 bp fragment of a part of N gene. All positive amplification products were confirmed by sequencing and compared with those in GenBank.</p><p><b>RESULTS</b>The overall frequency of coronavirus NL63 infection was 0.8%, 6 (60%) out of the coronavirus NL63 positive patients were detected in summer, 2 in autumn, 1 in spring and winter, respectively. The patients were from 2 months to two and a half years old. The clinical diagnosis was bronchopneumonia (60%), bronchiolitis (30%), and acute laryngotracheal bronchitis (10%). Four of the 10 cases had critical illness, 4 cases had underlying diseases, and 7 cases had mixed infection with other viruses. The homogeneity of coronavirus NL63 with those published in the GenBank at nucleotide levels was 97%-100%.</p><p><b>CONCLUSION</b>Coronavirus NL63 infection exists in hospitalized children with acute lower respiratory tract infection in Changsha. Coronavirus NL63 infections are common in children under 3 years of age. There is significant difference in the infection rate between the boys and the girls: the boys had higher rate than the girls. The peak of prevalence of the coronavirus NL63 was in summer. A single genetic lineage of coronavirus NL63 was revealed in human subjects in Changsha. Coronavirus NL63 may also be one of the lower respiratory pathogen in China.</p>

Viral etiology of 1165 hospitalized children with acute lower respiratory tract infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the viral etiology of acute low respiratory tract infection (ALRTI) among hospitalized children in Changsha of Hunan Province of China.</p><p><b>METHODS</b>Nasopharyngeal aspirates were collected from 1165 hospitalized children with ALRTI in Changsha from September 2007 to August 2008. Respiratory syncytin virus (RSV), human rhinovirus (HRV), influenza virus A (IFVA), influenza virus B (IFVB), parainfluenza 1-3 (PIV 1-3), human metapneumovirus (hMPV), human coronaviruses NL63 (HCoV-NL63), and human coronaviruses HKU1 (HCoV-HKU1) were detected by reverse transcription polymerase chain reaction (RT-PCR). Adenovirus (ADV) and human bocavirus (HBoV) were detected by standard polymerase chain reaction (PCR). WU polyomaviruses (WUPyV) and KI polyomaviruses(KIPyV) were detected by nested PCR. The positive samples further underwent genetic sequencing.</p><p><b>RESULTS</b>Among the 1165 nasopharyngeal aspirates, viruses were detected in 871 samples (74.76%), among which RSV (27.03%) was the most common virus, followed by HRV (17.33%), PIV3 (13.73%), HBoV (8.67%) and hMPV (6.52%). The overall positive rate of viral detection showed no significant differences between males and females (X2=2.241, P=0.134), whereas the positive rates of PIV3, hMPV, and HBoV in males were higher than in females. The positive rate of viral detection showed significant differences among different age groups (X2=10.934, P=0.027), and the highest positive rate was noted in the age group of 6 months to 1 year. Furthermore, the overall positive rate of viral detection showed a significant difference in term of seasonal distribution, with a peak prevalence in winter.</p><p><b>CONCLUSIONS</b>Virues predominate in the etiology of pediatric ALRTI in Changsha, and RSV, HRV and PIV3 are the main viruses for ALRTI. HBoV and hMPV have become increasingly important. Viral infection-associated ALRTI shows a prevail in the age group of 6 months to 1 year as well as in winter.</p>

Detection and clinical study on coronavirus HKU1 with acute lower respiratory tract infections of hospitalized children in Changsha / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In order to understand the epidemiological and virologic characteristics of coronavirus HKU1 infection in hospitalized children with acute respiratory tract infection (ARTI) in Changsha.</p><p><b>METHODS</b>1165 nasopharyngeal aspirates (NPA) specimens were collected from hospitalized children with ARTI between September 2007 and August 2008 in Changsha. Specimens were screened for pol gene of coronavirus HKU1 by polymerase chain reaction. All positive amplification products were confirmed by sequencing and compared with those in GenBank.</p><p><b>RESULTS</b>Coronavirus HKU1 were detected in 12 patients (1.03%) out of the 1165 children. The patients were from 8 days to 3 years. The most common clinical diagnosis was bronchopneumonia(83.33%). Similarity of coronavirus HKU1 with those published in the GenBank at nucleotide levels was 98.18% - 100%.</p><p><b>CONCLUSION</b>Coronavirus HKU1 may be important pathogens in children with acute lower respiratory tract infection. Coronavirus HKU1 infections are common in children under 3 years old. There is no significant difference in the infectious rate between the boys and the girls. The peak of its prevalence is in spring and winter. A single genetic lineage of Coronavirus HKU1 was revealed in human subjects in Changsha.</p>

Construction of human metapneumovirus DNA vaccine and study on its immune response in mice / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct human metapneumovirus (hMPV) DNA vaccines and evaluate the cellular and humoral immune response in mice.</p><p><b>METHODS</b>Fusion protein FdeltaTM (without transmembrane domain) gene and M gene of hMPV were amplified from cDNA by PCR, then DNA vaccines pcDNA3.1His-FdeltaTM and pcDNA3.1His-M were constructed to verify the expression of F and M protein by Western blotting and indirect immunofluorescent assay (IFA) respectively. Serum IgG and spleen cell CTL were detected with ELISA and ELISPOT assay after the BALB/c mice were immunized intramuscularly with the vaccines.</p><p><b>RESULTS</b>The candidate DNA vaccines could express FdeltaTM and M protein as detected with Western blotting and IFA. The IgG antibody titers of mice was 1:44 when immunized with pcDNA3.1His-FdeltaTM, but could increase to 1:64 when co-immunized with pcDNA3.1His-M. ELISPOT assay demonstrated that IFN-gamma-secreting effector T cells reached 42 +/- 8.9 in co-immunization group, higher than single vaccine pcDNA3.1His-FdeltaTM group (32 +/- 7.4).</p><p><b>CONCLUSION</b>DNA vaccine pcDNA3.1His-FdeltaTM could induce specific cellular and humoral immune responses, and the immune response could increase when co-immunization with pcDNA3.1His-M was carried out.</p>

Analysis of human metapneumovirus genotype in Hunan, China and its attachment protein G sequence character / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the genotypes of human metapneumovirus (hMPV) and the genetic character of hMPV attachment protein G sequence in Hunan, China.</p><p><b>METHODS</b>232 nasopharyngeal aspirates (NPA) samples from hospitalized children with acute respiratory infections were collected from Hunan, China in 2005. HMPV was detected. The full length of G glycoprotein genes were amplified and sequenced. Bioinformatics soft-wares were employed to analyze the sequences.</p><p><b>RESULTS</b>17/232 (7.3%) were showed hMPV positive. And co-infection rate with other viruses is 35%. The diagnoses of these hMPV positive cases are pneumonia, bronchiolitis and bronchopneumonia. Phylogenetic analysis for G genes from 13 hMPVs revealed the existence of four major subgroups: A1, A2, B1, B2 in Hunan, China in 2005. There are four types of sequence lengths of hMPV G glycoprotein, which are 711, 675, 660, 696nt. It is different in potential N-linked glycosylation sites and number of cysteine residues among these hMPVs of Hunan, China and Beijing, China. Also it is different from those in Japan and North America.</p><p><b>CONCLUSION</b>The investigation of hMPV from Hunan, China in 2005 revealed the high speed of genetic variation and the marked character of geographic epidemic differences.</p>

WU polyomavirus and KI polyomavirus detected in specimens from children with acute respiratory tract infection in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate newly identified polyomavirus WUV and WUV and KIPyV are associated with acute respiratory infections in China, tests were developed to detect WUV and KIPyV gene fragments from nasopharyngeal aspirates collected from children with ARI fron Nov. 2006 to Oct. 2007.</p><p><b>METHODS</b>A total of 318 clinical samples were tested for WUV and KIPyV using PCR method. The positive products were sequenced and compared with those in GenBank.</p><p><b>RESULTS</b>14 of the 318 Samples were positive (WUV was 2.2%, KIPyV was 2.2%). All of children who were positive for WUV or KIPyV had respiratory illness.</p><p><b>CONCLUSION</b>Polyomavirus WU and KIPyV infection may be associated with upper and lower respiratory diseases.</p>

Evaluation of reverse transcription loop-mediated isothermal amplification for detection of avian influenza A H5N1 virus / 病毒学报

A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.

Oxidative stress in patients with chronic hepatitis B before and after interferon alpha-2b treatment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the impacts of interferon alpha-2b (IFN alpha-2b) on the oxidative stress states in the treatment of chronic hepatitis B (CHB) with different genotypes.</p><p><b>METHODS</b>Thirty-five patients with chronic hepatitis B and 18 healthy volunteers as a control were enrolled in this present study. In control and patients group, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), serum malondialdehyde (MDA) levels, serum total antioxidative stress capacity (TAC) were measured spectrophotometrically. After the therapy with interferon alpha-2b at the dose of 300 million units via intramuscular injection thrice a week for 12 weeks, these parameters were measured again in the patient group. The genotypes of hepatitis B virus were detected by polymerase chain reaction and hybridization. The effective group was defined as the patients with complete response and partial response.</p><p><b>RESULTS</b>The elevated concentrations of MDA and impaired levels of TAC in the patients with CHB were observed as compared to the healthy controls (P < 0.05 for both). There were no significant differences in serum levels of MDA and TAC in CHB patients with various genotypes (P > 0.05). The serum levels of MDA after the treatment with IFN alpha-2b were significantly lower than the pretreatment levels (P < 0.05), which even returned to the normal concentration (P > 0.05) in the effective group. There were significant increases in the TAC after the IFN alpha-2b therapy in the effective group. However, the significant differences in the TAC levels before and after the INFalpha-2b treatment were not observed in the non-responsive group.</p><p><b>CONCLUSION</b>The oxidative stress could be improved with IFN alpha-2b treatment of chronic hepatitis B patients. The results suggest that antioxidant treatment for chronic hepatitis B patients may help improve the effect of anti-virus therapy.</p>

Expression, purification and antiviral activities of a new recombinant human interferon-lambda2 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To express recombinant human interferon lambda2 in E.coli and to study its antiviral activities.</p><p><b>METHODS</b>According to preferred codons used in E.coli, the highly-expressed human interferon lambda2 gene was designed, synthesized and cloned into expression vector pBV220 and transfected into E.coli DH5alpha. The expressed product was purified by using CM FF and size exclusion chromatography. Its antiviral activities were tested on different cells.</p><p><b>RESULTS</b>The expressed product was calculated about 15% of the total E.coli protein. The purified protein reached about 90% purity. Its specific antiviral activity was about 1.5 x 10(6) IU/mg on WISH/VSV test system. It was shown that the antiviral activity of the product on primates-origin cells seemed to be much higher than that on other non-primates-origin cells, indicating that interferon lambda2 possessed more stringent species specificity as compared with interferon-alpha2b. New interferon lambda2 showed similar anti-HBV activity as interferon-alpha2b.</p><p><b>CONCLUSION</b>Recombinant human interferon lambda2 could be expressed on E.coli. The purified product showed more stringent species specificity and similar anti-HBV activity as compared with interferon-alpha2b.</p>

Biological activities of recombinant human interferon Epsilon / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct a novel recombinant rhIFN-epsilon155ser, and study its biological activities.</p><p><b>METHODS</b>The whole sequence of rhIFN-epsilon was artificially synthesized and some codons were altered according to the preferred codon using of E.coli. The sequence was cloned into plasmid vector pBV220 to express in E.coli DH5alpha. After purification and re-folding of rhIFN-epsilon155ser inclusion body, the final product was tested for its biological activities, including anti-viral, anti-proliferative and NK cell enhancing activities. At the same time, by using DNA microarray biochips, the gene expression patterns in the rhIFN-epsilon155ser and rhIFN-alpha2b treated cells were compared and analyzed.</p><p><b>RESULTS</b>The re-built rhIFN-epsilon155ser sequence was expressed in E.coli as a form of inclusion body. After purified and re-folded, the rhIFN-epsilon155ser protein reached a purity of above 95%. The rhIFN-epsilon155ser protein had a specific anti-viral activity of about 6 x 10(5) IU/mg in WISH/VSV system. Its anti-proliferative activity and NK cell enhancing activities in vitro seemed to be lower than that of rhIFN-alpha2b. Data obtained from microarray biochips indicated that there were 283 pieces increasing 2 folds and 1489 pieces decreasing 2 folds among totally 22,278 pieces of human genes were found in the rhIFN-epsilon155ser treated cells; more changes in gene expression pattern were detected in the rhIFN-alpha treated cells.</p><p><b>CONCLUSION</b>A novel recombinant rhIFN-epsilon155ser was constructed, which belonged to type 1 interferon. The biological activities of rhIFN-epsilon155ser were compared with rhIFN-alpha2b. The changes of gene expression pattern in the interferon treated cells were detected, analyzed and discussed.</p>

Cloning, expression and purification of interferon-kappa, a novel human interferon, and its antiviral activity / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare human interferon-k (hIFN-kappa) and study its biological activities.</p><p><b>METHODS</b>Whole length of hIFN-kappa's cDNA was cloned, and its sequence was chemically synthesized according to the optimized codons of E.coli, then was expressed in E.coli DH5alpha. After purified, the rhIFN-kappa protein was tested for its various kinds of biological activities.</p><p><b>RESULTS</b>The purity of rhIFN-kappa was above 90%. In WHIS-VSV system, the antiviral activity of rhIFN-kappa was 2.0 x 10(6) IU/mg. Compared with rhIFN-alpha-2b, the biological activities of rhIFN-kappa were all feeble, including antiviral activity, promoting NK cell activity and anti-proliferation activity.</p><p><b>CONCLUSION</b>Antiviral activities of rhIFN-kappa on cell lines of different species are different, different viruses show different sensitivity to rhIFN-kappa.</p>

Preliminary study on nasal spray of interferon alpha-2b used for prevention of rubella and measles virus infections / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy of the interferon alpha-2b nasal spray in prevention of rubella and measles virus infections.</p><p><b>METHODS</b>The properly selected volunteer groups have been divided into interferon alpha-2b experimental and control group. The experimental group received interferon alpha-2b treatment by nasal spray for 2 days before the immunization, then both groups were challenged with rubella and measles attenuated live vaccine respectively through nasal spray. The sera from pre-immunization and 21 and 28 days after immunization were collected to test the IgG antibody titers. The influence on the viral antibody titer reflects the viral preventive effect by interferon alpha-2b.</p><p><b>RESULTS</b>The antibody titer difference of measles virus between experimental and control group was 1.26 (21 day) and 2.96 (28 day), there were statistically difference between them; the difference of rubella virus was 0.95 (21 day) and 0.37 (28 day), but there were no statistically differences found.</p><p><b>CONCLUSION</b>The preliminary results showed that the interferon alpha-2b can be used as prevention method for measles and rubella viral infections.</p>

A field trial of recombinant human interferon alpha-2b for nasal spray to prevent SARS and other respiratory viral infections / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the preventive effect of recombinant human interferon alpha-2b for nasal spray against SARS and other common respiratory viral infections by serum-epidemiological method.</p><p><b>METHODS</b>A randomized, placebo-controlled, double-blind field trial study in populations with 14,391 persons from SARS prevalent cities or provinces in China during May-Jun, 2003 and Dec-Apr, 2004. Interferon alpha-2b was given twice per day, once 9 x 10(5) IU by nasal spray for 5 days. Serum samples were taken at 15 days after last administration. Serological tests included SARS IgG antibody and IgM antibodies against influenza B, parainfluenza virus types 1-3, adenovirus type 3, 7 and respiratory syncytial virus by using commercial ELISA kits.</p><p><b>RESULTS</b>No statistically significant difference in serum SARS IgG antibody positive rate was found between the interferon and control groups among 2,757 serum samples. On the other hand, after using interferon, all four respiratory viruses (parainfluenza virus types 1-3 influenza B, adenovirus types 3, 7 and respiratory syncytial virus) in interferon group had lower IgM antibody positive rates than those in control group. Among them there were statistically significant differences between the interferon and control groups for parainfluenza virus, influenza B and adenovirus. The preventive efficacy of interferon against four respiratory viruses was different, from high to low, the rank was Flu B (66.76%), parainfluenza types 1-3 (66.75%), RSV (39.61%) and adenovirus (32.86%). The average preventive efficacy was 50.27%.</p><p><b>CONCLUSION</b>The recombinant human interferon alpha-2b for nasal spray could decrease the rates of common respiratory viruses infection in the selected population.</p>

A field trial for evaluating the safety of recombinant human interferon alpha-2b for nasal spray / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the safety of recombinant human interferon alpha-2b for nasal spray for the prevention of SARS and other upper respiratory viral infections.</p><p><b>METHODS</b>Field epidemiologic evaluation was conducted, the design was randomized and had a synchronously parallel control group. In the study, the drugs were given for five days and all subjects were followed up for ten days.</p><p><b>RESULTS</b>During the period of using interferon, body temperature of the experimental group was normal compared to the control group. Experimental group had more influenza-like symptoms than the control group (P < 0.05), such as headache (4.83%-7.09%), dizziness (7.17%-11.63%), lassitude (8.55%-15.06%), muscular soreness (4.43%-7.09%), pharynx dryness (12.10%-17.85%), angina (6.25%-8.72%), abdominal pain (2.30%-5.50%) and diarrhea (2.45%-5.66%). Most of side effects reached their peak with in the first 3 days. Except for pharynx dryness, the incidences of all other side effects declined after completion of the use of the trial drug, and incidences of some symptoms in experimental group were lower than those of the control group. There were no significant differences in the symptoms of cough and expectoration between the experimental group and the control group. The incidence of exanthem in the control group was significantly higher than that in the experimental group. The side effect of bloody nasal mucus was not observed in experimental group, which had been reported by other authors in several volunteer studies.</p><p><b>CONCLUSION</b>Using recombinant human interferon alpha-2b for nasal spray could lead to some influenza-like symptoms, however, all those symptoms were mild , reversible, and relieved after completion of the use of the trial drug. No serious side effects were found during the period of following up. The authors conclude that the drug is safe.</p>

Preventive and therapeutic effects of recombinant IFN-alpha2b nasal spray on SARS-CoV infection in Macaca mulata / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the preventive and therapeutic effects of recombinant IFN-alpha2b for nasal spray on SARS-CoV infection in Macaca mulata (rhesus monkey).</p><p><b>METHODS</b>Ten rhesus monkeys were divided into two groups, 5 in interferon group, and 5 in control group. Before and after SARS-CoV attack, the virus was detected in samples such as pharyngeal swab in all the two groups by Real-time PCR (RT-PCR) and virus isolation was performed.</p><p><b>RESULTS</b>After virus attack, the level of SARS-CoV-specific IgG and neutralizing antibody were induced by SARS-CoV in the interferon group was weaker than in control group. Hematology items showed no apparent changes after virus attack in treated group. Through pathological examination, the morphology of the lung tissues of two Macaques in the treated group was normal, while the other three displayed the interstitial pneumonia with the thickened septum and infiltration with mononuclear cells. Among which, one monkey showed part of thickened septum fused with each other. These lesions in the interferon treated animals were similar to those seen in the animals in control group, but with smaller scope of pathological changes. No significant abnormity was detected in other organs.</p><p><b>CONCLUSION</b>Recombinant IFN-alpha2b could effectively interdict or weaken SARS-CoV injury in monkeys.</p>

Cloning, expression of human keratinocyte growth factor and its purification and identification / 生物工程学报

To clone KGF-2 gene, get hKGF-2 protein and detemine its activity. The cNDA of human KGF-2 was isolated from fetal lung by RT-PCR and cloned into pBV220 plasmid. The recombinant pBV220-hKGF-2 plasmid was transformed into E. coli (BL21), induced at 42 degrees C for the expression of hKGF-2. Recombinant human KGF-2 was purified from the ultrasonic-treated BL21 by heparin-Sepharose CL-6B treated column chromatography and cation exchange column chromatography. MTT method was used for the determination of its biological activity. SDS-PAGE showed that rhKGF-2 was expressed in E. coli BL21 as soluble protein of approximately 20kD. The rhKGF-2 protein can stimulate the proliferation of NIH3T3 cells significantly from 1 ng/mL to 10 ng/mL. HKGF-2 cDNA wasclned and highly expressed in E. coli BL21 and the purified rhKGF-2 showed the mitogenic activity on NIH3T3 cells.

Producing human lactoferrin by high-density fermentation recombinant Pichia pastoris / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To evaluate expression of human lactoferrin gene by high-density fermentation in recombinant Pichia pastoris on the premise of maintaining its biological activities.</p><p><b>METHODS</b>The neutrophil was isolated from human peripheral blood and its total RNA was prepared. Full-length cDNA of human lactoferrin gene was then obtained by RT-PCR, cloned into expression vector pPIC 3.5 K and transformed into Pichia pastoris strain KM71. With two-layer filter method, the transformants with high-productivity of human lactoferrin were screened out into fed-batch high-density fermentation. And later, the physical, chemical and biological activities of fermentation product were detected preliminarily.</p><p><b>RESULTS</b>The strain p3.5-k-7 with better productivity of human lactoferrin was screened out into fed-batch high-density fermentation. The fermentation lasted nearly for nine days, with A-600 of culture once above 260 and the highest productivity of human lactoferrin being 115 mg/L, 7.67 times the amount of that in shake flask cultivation.</p><p><b>CONCLUSION</b>The authors successfully realized high-density fermentation expression of human lactoferrin gene in recombinant Pichia pastoris.</p>

Development of the cDNA chip for SARS virus and a primary study on the possible molecular mechanism of interferon alpha2b inhibiting the SARS virus replication / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the molecular mechanism of interferon alpha2b(IFNalpha2b) inhibiting the SARS virus replication. SARS-associated coronavirus (SARS virus) cDNA chip was developed and applied to detect the virus RNA transcription levels in the interferon-treated and untreated cell cultures, and the mechanism of anti-SARS virus activity of interferon alpha2b in cell culture system was explored.</p><p><b>METHODS</b>SARS virus cDNA chip was prepared by comparing the published SARS virus genome sequence, and the cDNA chip was used to study the interferon alpha2b function during SARS virus replication.</p><p><b>RESULTS</b>SARS virus cDNA chip was successfully prepared by using PCR method. The results showed that the cDNA chip could be used to detect the viral RNA transcription level. Interferon alpha2b could inhibit almost all the SARS virus gene transcription. An unknown gene at the position 28130-28426 bp, named as U gene, may play an important role during the viral replication.</p><p><b>CONCLUSION</b>A SARS virus whole genome cDNA chip was established. It could be used to study the virus molecular biology and antiviral drug screening. The results also showed that interferon alpha2b could inhibit almost the whole virus gene transcription by using the cDNA chip.</p>

Anti-SARS virus activities of different recombinant human interferons in cell culture system / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the anti-SARS virus activities of different recombinant human interferons on the cell culture system.</p><p><b>METHODS</b>Anti-SARS virus activities of interferons were determined by using CPE inhibition test in human skeletal muscle sarcoma (Rda) cell culture.</p><p><b>RESULTS</b>The average minimum amount of interferon alpha 2b, alpha 1b, beta 1b or omega 1b to inhibit 50% CPE in Rda cell culture was (160.5+/-129.5) IU/ml, (149.0+/-71.7) IU/ml, (69.5+/-61.5) IU/ml, (87.3+/-47.1) IU/ml, respectively or (0.6+/-0.5) ng/ml, (10.6+/-5.1) ng/ml, (3.5+/-3.1) ng/ml, (0.9+/-0.5) ng/ml, respectively.</p><p><b>CONCLUSION</b>All the tested recombinant interferons showed anti-SARS virus activities on the Rda cell culture with different sensitivities.</p>