Hepatitis B virus (HBV) is categorized into ten distinct genotypes (A - J), with over 40 subgenotypes identified to date. HBV genotype I (HBV-I), an inter-genotypic recombinant, has emerged during the evolution history of HBV. In this study, we conducted a comprehensive analysis of the genomic characteristics of HBV-I in China, employing a range of methodologies including phylogenetic analysis, nucleotide homology assessment, examination of amino acid substitutions within the PreS/S region, recombination detection, and evolutionary analysis. The 12 HBV-I strains, classified into subgenotype I1 and predominantly serotype adw2 (with one exception being ayw1) were preliminarily divided into two clusters based on homology analysis. A higher substitution rate was observed in the antigenic loop of the hepatitis B surface antigen (HBsAg), and the potential immune-escape mutations were found. Molecular clock analysis estimated an average evolutionary rate for HBV-I between 1.17 exp(-4) and 1.61 exp(-4) substitutions/site/year, with the most recent common ancestor traced back to between year 1740 and 1774. The epidemiological surveillance and genomic characterization of HBV genotype I are significant for informing future strategies in the prevention and control of hepatitis B.

To evaluate the long-term efficacy and anamnestic response of Chinese hamster ovary (CHO) cell-derived hepatitis B vaccine (CHO-HepB) after 18-20 years, a cross-sectional survey was conducted in seven communities in Zhengding County at the end of 2017. The birth cohort 1997-1999 vaccinated primarily with three doses of CHO-HepB were enrolled in the survey. The HBV serological markers were quantified using the Chemiluminescence method. The infection status of HBsAg-positive participants was determined by comparing their results with the previous data. For those with an anti-HBs antibody negative, the anamnestic response was evaluated by measuring anti-HBs antibody concentrations following a dose of HepB administration. A total of 1352 participants were enrolled, with the prevalence of HBsAg, anti-HBs, and anti-HBc being 0.4 %, 74.5 %, and 1.3 %, respectively. There was no significant difference in the HBV markers (HBsAg, anti-HBs and anti-HBc) between birth-year groups (P > 0.05). The geometric mean concentration (GMC) of anti-HBs antibodies among 1007 positive participants was 191 mIU/ml. No new infections or carriers were identified in the survey. Combined with the three previous surveys of the same birth cohort, the positive rates of HBsAg, anti-HBs, and anti-HBc remained largely unchanged over two decades following CHO-HepB vaccination. Of 248 participants who received a booster vaccination, 231 (93.1 %) showed an anti-HBs antibody positive with a GMC of 369 mIU/mL. Moreover, the positive rate and GMC of anti-HBs were higher in the CHO-HepB booster group compared to the Saccharomyces cerevisiae-HepB booster group. The long-term efficacy of the CHO-HepB remains stable for 18-20 years after primary vaccination, and a higher seroconversion rate of anti-HBs is observed following a booster vaccination of CHO-HepB. Given the absence of new infections or carriers over the past two decades, it appears unnecessary to administer a booster vaccination of HepB.

Objective Combination immunotherapy strategies targeting OX40, a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation, differentiation, and effector function of tumor-infiltrating T cells, have attracted much attention for their excellent therapeutic effects. In this study, we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core viruslike particles（HBc VLPs） therapy using a mouse colon cancer model.Methods Humanized B-h OX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-h OX40 antibody. Tumor growth was monitored. Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors.Results The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth,suggesting that a potent antitumor immunity was induced by the combination therapy. Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells（Teffs） and a significant decrease in regulatory T cells（Tregs） in the tumor microenvironment（TME）, which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs.Conclusion Combination therapy of anti-h OX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice, which may represent a potential design strategy for cancer immunotherapy.

Objective The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain (RBD) recombinant protein formulated with CpG 1826 plus alum dual adjuvant. Methods The BA.2 variant RBD (residues 308-548) fusing TT-P2 epitope was obtained from prokaryotic expression system, purification technology and dialysis renaturation, which was designated as Sot protein. The soluble Sot protein formulated with CpG 1826 plus alum dual adjuvant was designated as Sot/CA subunit vaccine and then the BALB/c mice were intramuscularly administrated with two doses of the Sot/CA subunit vaccine at 14-day interval (day 0 and 14). On day 28, the number of effector T lymphocytes secreting IFN-γ and IL-4 in mice spleen were determined by enzyme-linked immunospot (ELISpot) assay. The serum IgG, IgG1 and IgG2a antibodies were examined by enzyme-linked immunosorbent assay (ELISA). In addition, the level of neutralizing antibodies (NAbs) induced by Sot/CA subunit vaccine was also evaluated by the microneutralization assay. Results The high-purity soluble Sot protein with antigenicity was successfully obtained by the prokaryotic expression, protein purification and dialysis renaturation. The Sot/CA subunit vaccine induced a high level of IgG antibodies and NAbs, which were of cross-neutralizing activity against SARSCoV- 2 BA.2 and XBB.1.5 variants. Meanwhile, Sot/CA subunit vaccine also induced a high level of effector T lymphocytes secreting IFN-γ (635.00 ± 17.62) and IL-4 (279.20 ± 13.10), respectively. Combined with a decreased IgG1/IgG2a ratio in the serum, which indicating Sot/CA subunit vaccine induced a Th1-type predominant immune response. Conclusion The Sot protein formulated with CpG 1826 plus alum dual adjuvant showed that the excellent cellular and humoral immunogenicity, which provided a scientific basis for the development of BA.2 variant subunit vaccines and references for the adjuvant application of subunit vaccines.

Objective:A comparison of method for the detection of hepatitis E virus (HEV) in oysters was performed to provide a technical reference for the detection of HEV in oysters.Methods:After pre-treatment of oyster digestive gland specimens artificially contaminated with HEV fecal suspensions by the proteinase K digestion with reference to the European Union ISO/TS 15216-2∶2019, HEV RNA was extracted by four nucleic acid extraction method and assayed by Real time RT-PCR to compare the HEV recoveries; artificially contaminated oyster digestive gland specimens were pretreated by proteinase K digestion, proteinase K digestion + PEG precipitation, and proteinase K digestion + PEG precipitation + chloroform extraction, respectively, and HEV RNA was extracted by the optimal nucleic acid extraction method, which was assayed by real time RT-PCR to compare the HEV recoveries and inhibition rates of the three pretreatment method. The optimal HEV assay was applied to commercially available oyster specimens.Results:The HEV recoveries of the four nucleic acid extraction methods were 1.37%, 2.50%, 4.24% and 7.56%, respectively, with statistically significant differences ( F=847.220, P<0.001); The HEV recoveries for each of the three pre-treatment method were 6.02%, 13.65% and 21.17%, respectively, with statistically significant differences ( F=16.800, P<0.001), and the proteinase K digestion + PEG precipitation + chloroform extraction method had the highest recovery; the inhibition rates of the three method were 13.38%, 20.98% and 8.66%, respectively, and the differences were statistically significant ( F=20.205, P<0.001), with the lowest inhibition rate for the proteinase K digestion + PEG precipitation + chloroform extraction method. One HEV RNA positive specimen was detected in 120 commercially available oyster specimens. Conclusions:In the HEV detection of oyster specimens, pre-treatment with proteinase K digestion + PEG precipitation + chloroform extraction can improve the recovery of HEV from oysters and is more suitable for pre-treatment of oyster specimens; different manufacturers′ viral nucleic acid extraction method have different HEV recoveries and should be compared and screened for superiority before carrying out the assay.

Objective:To establish a digital droplet RT-PCR(dRT-PCR) method for Hepatitis A virus (HAV), and compare it with Real time RT-PCR(RT-qPCR) method, and select the best method for detecting hepatitis A virus in strawberry.Methods:Extract HAV vaccine RNA, optimize the reaction conditions of dRT-PCR and evaluate its specificity; Alkaline elution -PEG concentration method was used to extract nucleic acid from strawberry samples. At the same time, dRT-PCR and RT-qPCR method were used to detect the sensitivity and inhibition rate of HAV vaccine RNA in pure water and strawberry matrix, and the recovery rate of HAV in artificially contaminated strawberry was compared, which was applied to the detection of commercially available samples.Results:The optimal annealing temperature for dRT-PCR reaction was 60 ℃, and the optimal concentrations of primers and probes were 0.4 μmol/L、0.4 μmol/L and 0.2 μmol/L, with good specificity. There is no significant difference in sensitivity between the two method in detecting HAV vaccine RNA in pure water and strawberry matrix. The inhibition rate of dRT-PCR is low. The recovery rates of dRT-PCR and RT-qRCR in the detection of strawberry samples contaminated with HAV at higher concentrations were 12.90±0.006% and 30.12±0.02%, respectively. The recovery rates of lower concentrations of HAV contaminated strawberry samples were 18.27±0.07% and 10.85±0.03%, respectively, and the difference was statistically significant ( P<0.05). When strawberry samples on the market were tested, the result of both method were negative. Conclusions:The sensitivity of dRT-PCR method established in this study is not significantly different from that of RT-qPCR in detecting HAV RNA in different substrates, but dRT-PCR has good tolerance to PCR reaction inhibitors and high recovery rate when detecting low concentration HAV. Both detection method can be used for quantitative detection of hepatitis A virus in strawberry, and can be selected according to the actual situation.

Supplementary Table 1 from Distribution and Hepatocellular Carcinoma–Related Viral Properties of Hepatitis B Virus Genotypes in Mainland China: A Community-Based Study

Objective: To understand the distribution of genotypes and sub-genotypes of HBV in different ethnic groups in China. Methods: The HBsAg positive samples were selected by stratified multi-stage cluster sampling from the sample base of national HBV sero-epidemiological survey in 2020 for the amplification of S gene of HBV by nested PCR. A phylogeny tree was constructed to determine the genotypes and sub-genotypes of HBV. The distribution of genotypes and sub-genotypes of HBV were analyzed comprehensively by using laboratory data and demographic data. Results: A total of 1 539 positive samples from 15 ethnic groups were successfully amplified and analyzed, and 5 genotypes (B, C, D, I and C/D) were detected. The proportion of genotype B was higher in ethnic group of Han (74.52%, 623/836), Zhuang (49.28%, 34/69), Yi (53.19%, 25/47), Miao (94.12%, 32/34), Buyi (81.48%, 22/27). The proportions of genotype C were higher in ethnic groups of Yao (70.91%, 39/55). Genotype D was the predominant genotype in Uygur (83.78%, 31/37). Genotype C/D were detected in Tibetan (92.35%,326/353). In this study, 11 cases of genotype I were detected, 8 of which were distributed in Zhuang nationality. Except for Tibetan, sub-genotype B2 accounted for more than 80.00% in genotype B in all ethnic groups. The proportions of sub-genotype C2 were higher in 8 ethnic groups, i.e. Han, Tibetan, Yi, Uygur, Mongolian, Manchu, Hui and Miao. The proportions of sub-genotype C5 were higher in ethnic groups of Zhuang (55.56%, 15/27) and Yao (84.62%, 33/39). For genotype D, sub-genotype D3 was detected in Yi ethnic group and sub-genotype D1 was detected in both Uygur and Kazak. The proportions of sub-genotype C/D1 and C/D2 in Tibetan were 43.06% (152/353) and 49.29% (174/353). For all the 11 cases of genotype I infection, only sub-genotype I1 was detected. Conclusions: Five genotypes and 15 sub-genotypes of HBV were found in 15 ethnic groups. There were significant differences in the distribution of genotypes and sub-genotypes of HBV among different ethnic groups.

Supplementary Table 7 from Distribution and Hepatocellular Carcinoma–Related Viral Properties of Hepatitis B Virus Genotypes in Mainland China: A Community-Based Study

Rabies, caused by the rabies virus (RABV), is the most fatal zoonotic disease. It is a neglected tropical disease which remains a major public health problem, causing approximately 59,000 deaths worldwide annually. Despite the existence of effective vaccines, the high incidence of human rabies is mainly linked to tedious vaccine immunisation procedures and the overall high cost of post-exposure prophylaxis. Therefore, it is necessary to develop an effective vaccine that has a simple procedure and is affordable to prevent rabies infection in humans. RABV belongs to the genus Lyssavirus and family Rhabdoviridae. Previous phylogenetic analyses have identified seven major clades of RABV in China (China I-VII), confirmed by analysing nucleotide sequences from both the G and N proteins. This study evaluated the immunogenicity and protective capacity of SYS6008, an mRNA rabies vaccine expressing rabies virus glycoprotein, in mice and cynomolgus macaques. We demonstrated that SYS6008 induced sufficient levels of rabies neutralising antibody (RVNA) in mice. In addition, SYS6008 elicited strong and durable RVNA responses in vaccinated cynomolgus macaques. In the pre-exposure prophylaxis murine model, one or two injections of SYS6008 at 1/10 or 1/30 of dosage provided protection against a challenge with a 30-fold LD50 of rabies virus (China I and II clades). We also demonstrated that in the post-exposure prophylaxis murine model, which was exposed to lethal rabies virus (China I-VII clades) before vaccination, one or two injections of SYS6008 at both 1/10 and 1/30 dosages provided better protection against rabies virus challenge than the immunization by five injections of commercial vaccines at the same dosage. In addition, we proved that SYS6008-induced RVNAs could neutralise RABV from the China I-VII clades. Finally, 1/10 of the dosage of SYS6008 was able to stimulate significant RABV-G specificity in the T cell response. Furthermore, we found that SYS6008 induced high cellular immunity, including RABV-G-specific T cell responses and memory B cells. Our results imply that the SYS6008 rabies vaccine, with a much simpler vaccination procedure, better immunogenicity, and enhanced protective capacity, could be a candidate vaccine for post-exposure prophylaxis of rabies infections.

Rabies is a fatal zoonotic disease caused by the rabies virus. Despite existing vaccines, failures still persist. Complete protection relies on improving vaccination for delayed antibody response and weak cellular immunity. A more effective and secure vaccine is necessary for rabies prevention. For this purpose, we employed the use of PIKA adjuvant, a stabilized double-stranded RNA that interacts with TLR3, as an enhancer for the rabies immunization. Testing on mice infected with seven rabies strains prevalent in China showed over 80% protective efficacy without immunoglobulin. In contrast, the PIKA rabies vaccine exhibited a more significant enhancement in neutralizing antibody levels just 5 days post-vaccination, surpassing the immune response induced by licensed rabies vaccines. Furthermore, the administration of the PIKA rabies vaccine resulted in a significant augmentation in the population of T cells that produce IFN-γ in response to the antigen. Additionally, elevated levels of IL-1β, IL-6, CCL-2, and TNF-α were observed at the injection site. Furthermore, an increase in the levels of chemotactic proteins and pro-inflammatory molecules in the serum was observed following administration of the PIKA rabies vaccine. Confirmation of the mechanism of action of PIKA was further established by testing it on TLR3-knockout mice, proving that its adjuvant function is dependent on the TLR3 pathway. Taken together, these results indicate that the PIKA vaccine for rabies shows potential as a highly efficacious approach, resulting in a significant enhancement of the efficacy of rabies vaccines.

Protein cages have played a long-standing role in biomedicine applications, especially in tumor chemotherapy. Among protein cages, virus like particles (VLPs) have received attention for their potential applications in vaccine development and targeted drug delivery. However, most of the existing protein-based platform technologies are plagued with immunological problems that may limit their systemic delivery efficiency as drug carriers. Here, we show that using immune-orthogonal protein cages sequentially and modifying the dominant loop epitope can circumvent adaptive immune responses and enable effective drug delivery using repeated dosing. We genetically modified three different hepadnavirus core protein derived VLPs as delivery vectors for doxorubicin (DOX). These engineered VLPs have similar assembly characteristics, particle sizes, and immunological properties. Our results indicated that there was negligible antibody cross-reactivity in either direction between these three RGD-VLPs in mice that were previously immunized against HBc VLPs. Moreover, the sequential administration of multiple RGD-VLP-based nanomedicine (DOX@RGD-VLPs) could effectively reduce immune clearance and inhibited tumor growth. Hence, this study could provide an attractive protein cage-based platform for therapeutic drug delivery.

为分析具有相同VP1-2A区基因序列的甲型肝炎病毒(Hepatitis A virus,HAV)流行株全基因组序列特征,收集我国6个省市不同年份同一起或不同起甲型肝炎(甲肝)暴发中,部分甲肝病例急性期血清标本,提取HAV RNA,进行VP1-2A区基因分型,RT-PCR分段扩增HAV近全基因组序列,构建系统进化树,分析基因组特征.本研究获得16条HAV近全基因组序列,均属于基因ⅠA亚型,核苷酸和氨基酸序列同源性分别为95.81％～100％和99.23％～100％.与GenBank中HAV序列比较,15条序列与Man12-001(蒙古国株)最接近,核苷酸和氨基酸序列同源性分别为97.87％～99.81％和99.55％～99.95％;1条序列与HAJEF-K12(日本株)最接近,核苷酸和氨基酸序列同源性分别为99.37％和99.91％.本文中VP1-2A区序列完全相同的HAV流行株,来源于同一起甲肝暴发时,HAV近全基因组核苷酸序列差异为0％～0.03％;来源于不同起暴发时,核苷酸序列差异为0.18％～0.99％.16条HAV序列在已发表的中和抗原表位未发现变异,编码区氨基酸序列处于负向选择压力,16条序列间及与参考序列间未发现基因重组.本研究表明我国存在多株HAV流行株,主要为基因ⅠA亚型;VP1-2A区序列完全相同的HAV流行株,来源于同一起甲肝暴发的HAV全基因组核苷酸序列同源性高于不同起暴发的HAV序列同源性.HAV基因分型及全基因序列分析与现场流行病学调查结果相结合,更有利于HAV溯源分析.

目的 分析贵州省部分地区人源和猪源戊型肝炎病毒(Hepatitis E virus,HEV)流行株基因特征.方法 2019-2021年在贵州省医疗机构收集急性HEV感染者(抗-HEV IgM阳性)血清标本,在屠宰场收集猪胆汁和血清标本,提取HEV核酸,对HEV ORF2区基因进行扩增和测序,构建系统进化树,分析基因型、基因亚型及其核苷酸和氨基酸同源性.结果 从450例急性HEV感染者标本和196份猪标本中共获得53条人源和2条猪源HEV序列.人源序列的核苷酸和氨基酸同源性分别为81.39％-100％和95.92％-100％,猪源序列分别为87.40％和100％;人源和猪源HEV均与HEV基因4型参考株的核苷酸同源性最高,分别为89.93％和89.83％.55条HEV序列位于系统进化树的3个分支,其中24条与4b亚型、20条与4a亚型、11条与4d亚型参考株处于同一分支,且核苷酸同源性最高,分别为95.54％、93.10％、95.72％.2条人源序列各发生1个位点的氨基酸替换.结论 2019-2021年贵州省部分地区人群和猪群中HEV优势流行株为基因4型,包括4b、4a和4d亚型,氨基酸序列相对保守;可能存在跨种传播.

Although recognized as a curable disease, the persistence of hepatitis C virus (HCV) in chronically infected patients remains a great burden for public health. T cell immune responses serve a key role in anti-HCV infection; however, the features of T cell immunity in patients after a long-term infection are not well explored. We recruited a special cohort of patients with similar genetic background and natural developing progression of disease who were infected with HCV through blood donation 35 y ago. We found that self-resolved individuals had higher levels of cytokine-secreting T cells than individuals with chronic infections, indicating HCV-specific T cell immunity could be sustained for >35 y. Meanwhile, virus-specific CD8+ T cells in chronic patients were characterized by programmed cell death-1high, TIM-3high expression, which was related to liver injury characterized by aspartate transaminase/alanine aminotransferase levels and morphopathological changes. Unexpectedly, the expression of Lymphocyte-activation gene 3 on CD8+ T cells was lower in chronic patients and negatively correlated with alanine aminotransferase/aspartate transaminase. Our findings provided new insights into HCV-specific T cell responses and may shed light on a way to figure out novel effector targets and explore a way to reverse chronic infections.

[目的]原核表达非洲猪瘟病毒(African swine fever virus,ASFV)次要衣壳五邻体顶点蛋白(H240R),并研究其免疫原性.[方法]运用生物信息学软件初步预测分析H240R蛋白的理化性质、二级和三级结构.目的基因序列密码子优化后全基因合成,利用基因重组技术构建重组载体pET28 a/H,通过原核系统表达目的蛋白,筛选最优的诱导温度和诱导时间.目的蛋白(包涵体)通过含2M尿素洗涤缓冲液洗涤和亲和层析进行纯化.利用梯度透析对纯化的目的蛋白进行复性,Western和Dot-ELISA鉴定复性的目的蛋白.复性蛋白和佐剂ISA201乳化后免疫小鼠,评价H240R蛋白免疫原性及其在小鼠体内诱导的抗体消长规律.[结果]生物信息学分析显示H240R蛋白分子量约为27 kDa,等电点为9.40,无跨膜结构域,抗原指数较高.成功构建大肠杆菌工程菌株BL21(DE3)/pET28a/H,在不同温度诱导条件下H240R蛋白均以包涵体形式表达.包涵体蛋白经过洗涤和亲和层析纯化后,纯度达85％以上.大约65％纯化蛋白可通过梯度透析进行复性.Western显示His抗体可以和融合蛋白结合显色,表明目的蛋白表达正确,Dot-ELISA显示阳性血清和复性的目的蛋白反应,表明复性的H240R蛋白具有正确的构象.复性蛋白免疫小鼠后,可刺激小鼠产生抗体,二免后10 d达到平台期,可以稳定持续30 d左右.[结论]ASFV衣壳五邻体顶点蛋白H240R在原核系统中以包涵体形式表达,纯化复性的目的蛋白具有良好的免疫原性,并且可以和阳性血清反应.这些可为进一步研究H240R蛋白的结构和功能,以及ASFV亚单位疫苗研制提供参考和思路.

Without approved vaccines and specific treatments, COVID-19 is spreading around the world with above 26 million cases and approximately 864 thousand deaths until now. An efficacious and affordable vaccine is urgently needed. The Va1308 - Gly548 of spike protein of SARS-CoV-2 linked with Gln830 - Glu843 of Tetanus toxoid (TT peptide) (designated as S1-4) and without TT peptide (designated as S1-5) were expressed and renatured. The antigenicity and immunogenicity of S1-4 were evaluated by Western Blotting (WB) in vitro and immune responses in mice, respectively. The protective efficiency was measured preliminarily by microneutralization assay (MN50). The soluble S1-4 and S1-5 protein was prepared to high homogeneity and purity. Adjuvanted with Alum, S1-4 protein stimulated a strong antibody response in immunized mice and caused a major Th2-type cellular immunity supplemented with Th1-type immunity. Furthermore, the immunized sera could protect the Vero E6 cells from SARS-CoV-2 infection with neutralizing antibody titer 256. Recombinant SARS-CoV-2 RBD with a built in T helper epitope could stimulate both strong humoral immunity supplemented with cellular immunity in mice, demonstrating that it could be a promising subunit vaccine candidate. (C) 2021 Elsevier Ltd. All rights reserved.

SARS-CoV-2 is the cause of the worldwide outbreak of COVID-19 that has been characterized as a pandemic by the WHO. Since the first report of COVID-19 on December 31, 2019, 179,111 cases were confirmed in 160 countries/regions with 7426 deaths as of March 17, 2020. However, there have been no vaccines approved in the world to date. In this study, we analyzed the biological characteristics of the SARS-CoV-2 Spike protein, Pro330-Leu650 (SARS-CoV-2-SPL), using biostatistical methods. SARS-CoV-2-SPL possesses a receptor-binding region (RBD) and important B (Ser438-Gln506, Thr553-Glu583, Gly404-Aps427, Thr345-Ala352, and Lys529-Lys535) and T (9 CD4 and 11 CD8 T cell antigenic determinants) cell epitopes. High homology in this region between SARS-CoV-2 and SARS-CoV amounted to 87.7%, after taking the biological similarity of the amino acids into account and eliminating the receptor-binding motif (RBM). The overall topology indicated that the complete structure of SARS-CoV-2-SPL was with RBM as the head, and RBD as the trunk and the tail region. SARS-CoV-2-SPL was found to have the potential to elicit effective B and T cell responses. Our findings may provide meaningful guidance for SARS-CoV-2 vaccine design.

促吞噬肽(Tuftsin)是机体脾组织产生的生理活性肽,具有强大的免疫调节和免疫治疗潜力.乙型肝炎病毒核心蛋白病毒样颗粒(hepatitis B virus core protein virus-like particles,HBc VLPs)是由HBc自组装形成的空心纳米颗粒,其不仅能应用于药物的递送,还能应用于外源蛋白质的显示.因此,Tuftsin功能化HBc VLPs载体的研究在免疫治疗、分子递送等方面具有重要意义.本研究选用PET43.1-a质粒作为Tuftsin-HBc VLP的表达载体,以大肠杆菌BL21 (DE3)为工程菌进行诱导表达,通过盐析、分子筛层析和离子交换层析技术纯化生产Tuftsin-HBc VLP.利用Western印迹和ELISA分别对Tuftsin-HBc VLP上HBc和Tuftsin进行定性分析.结果 显示,Tuftsin-HBc VLP可与抗HBc抗体和抗Tuftsin抗体发生特异性结合.透射电镜观察结果显示,Tuftsin-HBc VLP呈大小均一的球形结构,粒度分析仪测得Tuftsin-HBc VLP的直径约为30 nm.CCK8法显示,Tuftsin-HBc VLP在0～ 480 μg/mL范围内,细胞增殖未见显著变化.上述结果表明,本研究成功制备了可以在原核系统中高效表达且安全有效的Tuftsin-HBc VLP生物纳米递送载体.

西藏地区藏族人群乙型肝炎病毒(Hepatitis B virus,HBV)感染率较高,而针对感染者血清中HBV表面抗原(Hepatitis B surface antigen,HBsAg)和HBV表面抗原抗体(Hepatitis B surface antibody,HBsAb)双阳性的研究一直进展缓慢,尚无明确的研究结论.为探讨西藏地区藏族人群慢性HBV感染者血清中HBsAg和HBsAb双阳性与基因组核苷酸/氨基酸突变的关系,本研究在西藏选取7个地区作为研究区域,进行多阶段抽样,选取样本进行HBV血清五项指标检测,筛选HBsAg和HBsAb均为阳性的患者血清共24份作为双阳性组,以年龄和乙型肝炎e抗原(HBeAg)等感染指标进行匹配,选取96份HBsAg阳性,HBsAb阴性患者血清作为对照组.HBV全基因组序列通过聚合酶链式反应(Polymerase chain reaction,PCR)产物直接测序获得,并进行重组分析和突变分析.852名西藏HBV感染者中,HBsAg/HBsAb双阳性率为2.82％(24/852).双阳性组在S蛋白N端和主要亲水区(Major hydrophilic region,MHR)的突变率以及PreS缺失发生率均显著高于对照组.T1753C、C1990T和C2002T等核苷酸突变;S蛋白中V224A 、PreS区D103E等氨基酸突变在两组内分布存在显著差异.HBV/CD重组型的HBsAg/HBsAb双阳性发生率与中国乙肝主要流行区域接近.HBV感染者血清HBsAg和HBsAb共存可能与S蛋白,特别是MHR内的高氨基酸突变造成的免疫逃逸有关.PreS缺失、S抗原蛋白C端V224A突变和PreS区D103E突变可能对HBsAg/HBsAb双阳性的产生具有协同作用.