Innate immune sensing of self-derived double-stranded RNA by RIG-I-MAVS-TNF-α regulates the survival and senescence fate of SARS-2-S syncytia

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains an important health threat. Syncytial formation by infected cells mediated by the SARS-CoV-2 spike protein (SARS-2-S) is a hallmark of COVID-19-associated pathology. Although SARS-CoV-2 infection evokes cellular senescence, as in other viruses, the direct link between SARS-2-S-induced syncytia with senescence in the absence of viral infection and their senescence fate determinants remain unknown. Here, we show that syncytia formed by cells expressing exogenously delivered SARS-2-S exhibited a senescence-like phenotype in vitro and that SARS-2-S mRNA induced senescence phenotype in vivo. Extracellular vesicles (EVs) containing SARS-2-S also induced senescent syncytium formation independent of the de novo synthesis of SARS-2-S. Mechanistically, we show that the accumulation of endogenous dsRNA, partially that whose formation is induced by activation of the unfolded protein response (UPR), in SARS-2-S syncytia triggers RIG-I-MAVS signalling to drive the TNF--dependent survival and senescence fate of SARS-2-S syncytia. Our findings suggest that the fusogenic ability of SARS-2-S might contribute to the side effects of particular COVID-19 vaccines or perhaps long COVID-19 syndrome and provide insight into how these effects can be prevented.

Antibody Persistence and Safety through 6 Months after Heterologous Orally Aerosolised Ad5-nCoV in individuals primed with two-dose CoronaVac previously

BackgroundHeterologous orally administered adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in individuals who were primed with two-dose CoronaVac (an inactivated SARS-CoV-2 vaccine, by Sinovac) previously, has been reported to be safe and highly immunogenic within 28 days post-boosting. However, antibody persistence and safety up to 6 months of this regimen are not been reported yet. MethodsThis is a randomized, open label, single-center trial on safety and immunogenicity of heterologous boost immunization with an orally administered aerosolised Ad5-nCoV vs. homologous boost immunization with CoronaVac after two-dose priming with CoronaVac in Chinese adults aged 18 years and older (NCT05043259). We followed the participants in this trial, including 140 in the low-dose aerosolised Ad5-nCoV group, 139 in the high-dose aerosolised Ad5-nCoV group, and 140 in the CoronaVac group for 6 months. Neutralising antibodies (NAbs) against live wild-type SARS-CoV-2 virus and omicron variant, and receptor-binding domain (RBD)-specific IgG antibodies were detected in serum samples collected at 28 days, 3 months, and 6 months after the booster dose. Serious adverse events (SAEs) were documented till month 6. ResultsThe low-dose and high-dose heterologous boost immunisation groups had NAb GMTs against live wild-type SARS-CoV-2 of 1937.3 [95% CI 1466.9, 2558.4] and 1350.8 [95% CI 952.6, 1915.3], which were 26.4 folds and 18.4 folds higher than that the CoronaVac group did (73.5 [95% CI 52.3, 103.3]) at 28 days. The low-dose and high-dose heterologous boost immunisation groups had NAb GMTs against live wild-type SARS-CoV-2 of 530.1 (95% CI 412.5, 681.1) and 457.6 (95%CI 349.4, 599.2), which were 26.0 folds and 22.4 folds higher than that the CoronaVac group did (20.4 [95%CI 14.3, 29.1]) at 3 months, respectively. At 6 months, the low-dose and high-dose heterologous booster groups had NAb GMTs against live wild-type SARS-CoV-2 of 312.9 (95% CI 237.7, 411.8) and 251.1 (95% CI 178.2, 354.0), which were 30.1 folds and 24.1 folds higher than the CoronaVac group did (10.4 [95% CI 7.8, 14.0]), respectively. Additionally, the low-dose and high-dose heterologous booster groups had NAb GMTs against live omicron variant of 52.0 (95% CI 37.2, 72.6) and 23.1 (95% CI 15.7, 33.9) at 28 days, 27.9 (95% CI 18.8, 41.3) and 23.3 (95% CI 16.2, 33.3) at 3 months, 16.0 (95% CI 10.9, 23.5) and 12.0 (95% CI 8.5, 16.8) at 6 months, respectively. However, nearly all participants had no detectable NAbs for omicron variant in the CoronaVac group at either 28 days, 3 months, or 6 months. No vaccine-related SAEs were observed. ConclusionsThese data suggested that heterologous aerosolised Ad5-nCoV following two-dose CoronaVac priming was safe and persistently more immunogenic than three-dose CoronaVac, although immune responses waned over time.

Heterologous CoronaVac plus Ad5-nCOV versus homologous CoronaVac vaccination among elderly: a phase 4, non-inferiority, randomized study

ImportancePeople over 60 developed less protection after two doses of inactivated COVID-19 vaccine than younger people. Heterologous vaccination might provide greater immunity and protection against variants of concern. ObjectiveTo assess the safety and immunogenicity of a heterologous immunization with an adenovirus type 5-vectored vaccine (Convidecia) among elderly who were primed with an inactivated vaccine (CoronaVac) previously. DesignAn observer-blind, randomized (1:1) trial, conducted from August 26 to November 13, 2021. SettingA single center in Jiangsu Province, China. Participants299 participants aged 60 years and older, of them 199 primed with two doses of CoronaVac in the past 3-6 months and 100 primed with one dose of CoronaVac in the past 1-2 months. InterventionConvidecia or CoronaVac as boosting dose Main Outcomes and MeasuresGeometric mean titers (GMTs) of neutralizing antibodies against wild-type SARS-CoV-2, and Delta and Omicron variants 14 days post boosting, and adverse reactions within 28 days. ResultsIn the three-dose regimen cohort (n=199; mean (SD) age, 66.7 (4.2) years; 74 (37.2%) female), 99 and 100 received a third dose of Convidecia (group A) and CoronaVac (group B), respectively. In the two-dose regimen cohort (n=100; mean (SD) age, 70.5 (6.0) years; 49 (49%) female), 50 and 50 received a second dose of Convidecia (group C) and CoronaVac (group D), respectively. GMTs of neutralizing antibodies against wild-type SARS-CoV-2 at day 14 were 286.4 (95% CI: 244.6, 335.2) in group A and 48.2 (95% CI: 39.5, 58.7) in group B, with GMT ratio of 6.2 (95% CI: 4.7, 8.1), and 70.9 (95% CI: 49.5, 101.7) in group C and 9.3 (95% CI: 6.2, 13.9) in group D, with GMT ratio of 7.6 (95% CI: 4.1, 14.1). There was a 6.3-fold (GMTs, 45.9 vs 7.3) and 7.5-fold (32.9 vs 4.4) increase in neutralizing antibodies against Delta and Omicron variants in group A, respectively, compared with group B. However, there was no significant difference between group C and group D. Both heterologous and homologous booster immunizations were safe and well tolerated. Conclusions and RelevanceHeterologous prime-boost regimens with CoronaVac and Convidecia induced strong neutralizing antibodies in elderly, which was superior to that induced by the homologous boost, without increasing safety concerns. Trial RegistrationClinicalTrials.gov NCT04952727 Key Points QuestionDoes a heterologous immunization with recombinant adenovirus type 5-vectored vaccine (Convidecia) produced a non-inferior or superior response of neutralizing antibodies among elderly primed with two doses of inactivated COVID-19 vaccine (CoronaVac), compared to the homologous boosting FindingsIn this randomized clinical trial, a heterologous third dose of Convidecia resulted in a 6.2-fold (geometric mean titers: 286.4 vs 48.2), 6.3-fold (45.9 vs 7.3) and 7.5-fold (32.9 vs 4.4) increase in neutralizing antibodies against wild-type strain, Delta and Omicron variants 14 days post boosting, respectively, compared to the homologous boost with CoronaVac MeaningHeterologous prime-boost regimens with CoronaVac and Convidecia induced strong neutralizing antibodies in elderly, which was superior to that induced by the homologous boosting.

Aerosolized Ad5-nCoV booster vaccination elicited potent immune response against the SARS-CoV-2 Omicron variant after inactivated COVID-19 vaccine priming

The SARS-CoV-2 Omicron variant has become the dominant SARS-CoV-2 variant around the world and exhibits immune escape to current COVID-19 vaccines to some extent due to its numerous spike mutations. Here, we evaluated the immune responses to booster vaccination with intramuscular adenovirus-vectored vaccine (Ad5-nCoV), aerosolized Ad5-nCoV, a recombinant protein subunit vaccine (ZF2001) or homologous inactivated vaccine (CoronaVac) in those who received two doses of inactivated COVID-19 vaccines 6 months prior. We found that the Ad5-nCoV booster induced potent neutralizing activity against the wild-type virus and Omicron variant, while aerosolized Ad5-nCoV generated the greatest neutralizing antibody responses against the Omicron variant at day 28 after booster vaccination, at 14.1-fold that of CoronaVac, 5.6-fold that of ZF2001 and 2.0-fold that of intramuscular Ad5-nCoV. Similarly, the aerosolized Ad5-nCoV booster produced the greatest IFN{gamma} T-cell response at day 14 after booster vaccination. The IFN{gamma} T-cell response to aerosolized Ad5-nCoV was 12.8-fold for CoronaVac, 16.5-fold for ZF2001, and 5.0-fold for intramuscular Ad5-nCoV. Aerosolized Ad5-nCoV booster also produced the greatest spike-specific B cell response. Our findings suggest that inactivated vaccine recipients should consider adenovirus-vectored vaccine boosters in China and that aerosolized Ad5-nCoV may provide a more efficient alternative in response to the spread of the Omicron variant.

Heterologous prime-boost immunization with CoronaVac and Convidecia

BackgroundThe safety and immunogenicity of heterologous prime-boost COVID-19 vaccine regimens with one shot of a recombinant adenovirus type-5-vectored COVID-19 vaccine Convidecia has not been reported. MethodsWe conducted a randomized, controlled, observer-blinded trial of heterologous prime-boost immunization with CoronaVac and Convidecia in healthy adults 18-59 years of age. Eligible participants who were primed with one or two doses of CoronaVac were randomly assigned at a 1:1 ratio to receive a booster dose of Convidecia or CoronaVac. Participants were masked to the vaccine received but not to the three-dose or two-dose regimen. The occurrences of adverse reactions within 28 days after the vaccination were documented. The geometric mean titers of neutralizing antibodies against live SARS-CoV-2 virus were measured at 14 and 28 days after the booster vaccination. ResultsBetween May 25 and 26, 2021, a total of 300 participants were enrolled. Participants who received a booster shot with a heterologous dose of Convidecia reported increased frequencies of solicited injection-site reactions than did those received a homogeneous dose of CoronaVac, but frequencies of systemic reactions. The adverse reactions were generally mild to moderate. The heterologous immunization with Convidecia induced higher live viral neutralizing antibodies than did the homogeneous immunization with CoronaVac (197.4[167.7, 232.4] vs. 33.6[28.3, 39.8] and 54.4[37. 9, 78.0] vs. 12.8[9.3, 17.5]) at day 14 in the three- and two-dose regimen cohort, respectively. ConclusionsThe heterologous prime-boost regimen with Convidecia after the priming with CoronaVac was safe and significantly immunogenic than a homogeneous boost with CoronaVac (ClinicalTrials.gov, number NCT04892459).

A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.

Detection of serum homocysteine levels in retinal branch vein occlusion patients with hypertension or non-hypertension / 中华眼底病杂志

Objective To observe the serum homocysteine (Hcy) levels in retinal branch vein occlusion (BRVO) patients with with hypertension or non-hypertension.Methods A total of 120 patients (120 eyes) with BRVO were divided into hypertension group [72 eyes,blood pressure 140-175/90-105 mmHg (1 mmHg=0.133 kPa)] and non-hypertension group (48 eyes,blood pressure 100-139/70-88 mmHg).According to the sex and age,78 patients with hypertensive non-retinal vascular diseases and 48 patients with non-hypertensive and non-retinal vascular diseases were collected by a way of same-size ratio as hypertension control group and non-hypertension control group,respectively.Fasting venous blood was collected from all patients in the moming and serum Hcy levels were measured by rate method.The total Hcy concentration over 15.0 μ mol/L was defined as high level Hcy.Fasting serum glucose and fasting serum lipid were also measured.Measurement data among groups were compared with t test.Results The serum Hcy levels were (26.82 ± 28.0),(8.39± 3.11),(21.37 ± 4.24),(9.25 ± 3.31) μmol/L in the hypertension group,hypertension control group,non-hypertension group and non-hypertension control group,respectively.The serum Hcy levels of patients in the hypertension group was significantly higher than that in the hypertension control group (t=3.324,P=0.004).The serum Hcy levels of patients in the non-hypertension group was significantly higher than that in the non-hypertension control group (t=2.216,P=0.049).The serum Hcy levels of patients in the hypertension group was significantly higher than that in the non-hypertension group,but the difference had not statistical significance (t=0.581,P=0.566).Among 120 patients,there were 68 patients (56.67％) with high level of Hcy (40 patients in the hypertension group and 28 patients in the non-hypertension group).Among the 40 patients with high levels of Hcy in the hypertension group,36 patients were older than 50 years old (90.00％) and 4 patients were less or equal than 50 years old (10.00％).Among the 28 patients with high levels of Hcy in the non-hypertension group,16 patients were older than 50 years old (57.14％);12 patients were less or equal than 50 years old (42.86％),whose indexes of serum glucose and serum lipid were not abnormal.There was significant difference in age distribution of patients with high level of Hcy between the hypertension group and the non-hypertension group (x2=9.882,P=0.002),but there was no significant difference in sex distribution (x2=2.052,P=0.216).Conclusions The level of serum Hcy increased both in BRVO patients with hypertension and non-hypertension.The indexes of serum glucose and serum lipid were not abnormal in BRVO patients aged less or equal than 50 years old with non-hypertensive except for the increase of serum Hcy level.

The imaging diagnosis of synovial chondromatosis of the temporomandibular joint / 实用放射学杂志

Objective To investigate X‐ray ,CT and MRI features of synovial chondromatosis of the temporomandibular joint (TMJ) .Methods X‐ray ,CT and MRI features of eight patients of synovial chondromatosis of TMJ with histo‐pathologically con‐firmed were analyzed retrospectively .X‐ray examination and CT scanning were performed in all eight patients .Routine MRI scanning was performed in six patients and contrast‐enhanced MRI scanning was performed in two patients synchronously .Results Tumors occured unilaterally in all eight cases ,which occured on the right TMJ in six cases and on the left side in two cases .On X‐ray films , widen joint space and calcificated loose bodies occured in all eight cases .On CT scanning ,cystic‐solid mixed mass around the joint and calcificated loose bodies occured in all eight cases .On MR scanning ,multiple nodular long T1 and short T2 signal occured in six cases . Arthroedema and synovial hyperplasia with iso T1 and iso or slightly long T2 signal in six cases .On contrast‐enhanced MR ,homoge‐neous enhancement occurred in svnovial tissue and the edge of loose bodies in two cases .Conclusion The synovial chondromatosis of TMJ owns typical imaging features .The imaging findings can serve as a reference to improve diagnosis of synovial chondromatosis of TMJ .

Three-dimensional Constructive Interference Steady State Sequence in Evaluation Dorsal Root Ganglion Compression in Lumbar Disc Herniation / 中国医学影像学杂志

Purpose To investigate the diameter change of dorsal root ganglion (DRG) in lumbar disc herniation using three-dimensional MR neurography. Materials and Methods Sixty-ifve patients with lumbar disc herniation and 30 healthy volunteers were selected. Bilateral DRG diameter was measured using MR three-dimensional constructive interference steady state (3D-CISS) sequence at the level of L3-S1 in the control group and at the level of herniation disc in patient group including central and lateral subgroups. The relationship between the sagittal index and DRG diameter at the level of herniation disc was analyzed. Results In the control group, the DRG diameters increased from the level of L3 to S1. The DRG diameters of the central subgroup were bigger than those of the control group (t=-2.485--2.253, P0.05). The sagittal index was not correlated with DRG diameter. Conclusion 3D-CISS sequence clearly demonstrates morphological changes of lumbosacral nerve root and measures its diameter.

Production and immunogenicity analysis of conformation-stable fragment-C mutant of tetanus toxin / 生物工程学报

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to A1a and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to > 95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1 x 10(3) 50% lethal doses (LD50s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.

Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase / 生物工程学报

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.

Establishment of a Mouse Model of Human PSCA-Expressing Prostate Cancer / 中国实验动物学报

Objective To establish a mouse model of prostate cancer expressing human PSCA for the development of new anti-tumor drugs or vaccines. Methods The total RNA of DU145 cells,a human prostate cancer cell line,was isolated by using TRIzol reagent according to the (RT-PCR),the first-strand cDNA was synthesized using the SuperScript First-Strand synthesis system. The human PSCA gene was amplified with the primers and cloned into the plasmid pcDNA3.1 to generate pcDNA-PSCA. DNA sequencing was used to confirm the constructs. The mouse prostate tumor cell line RM-1 cells,syngeneic to C57BL/6,were transfected with pcDNA-PSCA plasmids followed by selection using G418. RT-PCR analysis was performed to examine the validity of the constructs. Expression of PSCA on the cell surface was determined by staining with anti-PSCA antibody,and the anti-PSCA antibody was detected using an FITC-conjugated goat anti-rabbit IgG antibody,and analyzed by flow cytometry. 4-6-week-old male C57BL/6 mice purchased from the Laboratory Animals Center were inoculated with different amounts of RM-PSCA cells to search for suitable cell population which can form tumor in mouse,and the mice were monitored twice a week. The growth and the survival time of mice were measured,respectively. The tumor volume was measured by vernier caliper according to the formula:V=0.5a×b~2,where a and b are the long and short diameters of the tumor,respectively. Results The plasmid pcDNA-PSCA was successfully constructed and the PSCA was successfully expressed in RM-PSCA 7~# and RM-PSCA 28~# cells by RT-PCR and confirmed by flow cytometry. 1×10~5 RM-PSCA cells were sufficient to get tumor growth in 100% of inoculated mice. The tumor grew quickly and the volume of the tumor reached 12000 mm~3 within 34 days. All the mice died within 40 days and their mean survival time was 37 days. Conclusion A PSCA-expressing tumor model in mice has been successfully established. It can be used to evaluate the activities of drugs or vaccines.

Expression and biological activity analysis of human-mouse chimeric antibody against anthrax protective antigen / 中华微生物学和免疫学杂志

Objective To express human-mouse chimeric antibody against anthrax protective anti-gen and to analyze its biological activities. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reduetase(DHFR) gene as the selection and complication marker. First, the light and heavy chain variable region gene of the monoclonal antibody 5E1 were cloned by RT-PCR, at the same time the human IgG1 heavy chain constant region gene and kappa type constant region gene were cloned. Next, the human-mouse chimeric antibody genes were synthesized by fusion PCR. Then, the hu-man-mouse chimeric antibody gene were inserted into MCS of pSecTag and B1 to construct pSecTag-5E1L and B1-5E1H, respectively. Finally, heavy chain expression cassette excised from the B1-5E1H with Bgl Ⅱ/BamH Ⅰ was further cloned into the Bgl Ⅱ site of the pSecTag-5E1L to construct pSecTag-5E1. Plasmid pSecTag-5E1 was transfected into CHO(dhfr) engineering cells and high production cell clones that were screened by enhancing MTX concentration. After collecting medium and purifying chimeric antibody with af-finity chromatogram, purified chimeric antibody was analyzed by SDS-PAGE, Western blot. Results A sta-ble and high production cell line was acquired at MTX concentration 5×10~(-8) mol/L. Conclusion The hu-man-mouse chimeric antibodies were successfully expressed in CHO cells.

The SARS-CoV 3a and 7a Protein May Enhance the Induction of IFN-? / 中国生物工程杂志

3a and 7a are nonstructural proteins of SARSCoV, which are encoded separately by ORF 3a and ORF 7a in SARSCoV genome. The expression of 3a has been founded in cells infected by virus in vivo or in vitro. Firstly, the pGL3Control vector was reconstructed , the pGL3Enhancer vector deletious of SV40 promoter gene was obtained . Then the IFN? promoter gene was cloned into the pGL3Enhancer vector and pGLIP21, the Luciferase reporter plasmid with IFN? promoter was established. The availability of pGLIP21 was verified by NDV ,the inductor of IFN?, the Luciferase activity was assayed in cells transfected with pGLIP21 by Luminometer. In order to see the function of 3a and 7a protein of SARSCoV,CHO cells expressing 3a or 7a protein were transfected with pGLIP21, the intensity of luciferase activity was analyzed . By analysis, in vitro, 3a and 7a protein of SARSCoV had the similar ability in triggering the expression of Luceferase gene, i.e 3a and 7a protein of SARSCoV could effectively activate the promoter fragment of IFN? gene. This result will help studying the function of 3a and 7a protein and provide a method to study the nosogenesis mechanism of SARSCoV.

Expression and characterization of hepatitis C virus NS3 protease domain in insect cells / 中华传染病杂志

Objective To produce the recombinant NS3 protease of the hepatitis C virus in insect cells. Methods The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmid had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmid DNA was verified to replicate in insect cells and package into baculovirus particles via PCR and electronic microscope analysis. The insect cells infected by recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-Lauryl Sarcosine Sodium (NLS) and purifed by metal-chelated-affinity chromatography, then the enzymatic activity was measured with the protein substrate NS5ab and the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbent assay. Results The HCV NS3 protease domain was expressed in insect cells at high level and it was partially resolved in NLS;Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5?10 7 cells and both enzymatic activity and antigenicity of the protein were proven to be good. Conclusion The recombinant HCV NS3 protease expressed in insect cells is a membrane-binding protein with good enzymatic activity and antigenicity.