人乳头瘤病毒16型晚期蛋白L1的表达及病毒样颗粒的装配

目的　在昆虫细胞中表达人乳头瘤病毒 16型 (HPV16 )哈尔滨地区分离株的晚期蛋白L1,并分离纯化由L1蛋白在细胞中自主组装成的病毒样颗粒 ,为HPV16预防性疫苗的研制奠定基础。方法　获得含有HPV16L1基因的重组杆状病毒并使目的蛋白L1在昆虫细胞中表达 ;对重组杆状病毒的扩增条件及L1蛋白的表达水平进行优化 ;电镜下观察昆虫细胞中晚期蛋白装配成病毒样颗粒的情况 ,经氯化铯密度梯度纯化病毒样颗粒 ,并经Westernblot进行验证。结果　获得了稳定表达L1蛋白的重组杆状病毒 ;以MOI值为 0 .2感染昆虫细胞可获得较高滴度的重组病毒 ,以MOI值为 10感染昆虫细胞可获得较高水平的L1蛋白表达 ;电镜下可观察到昆虫细胞核内直径为 5 5nm的病毒样颗粒 ;病毒样颗粒可通过氯化铯密度梯度离心获得。结论　获得人乳头瘤病毒哈尔滨地区分离株L1蛋白可在昆虫细胞中自主装配的病毒样颗粒并成功分离纯化。     

原核高效制备人乳头瘤病毒16型L1病毒样颗粒

目的通过原核表达系统高效制备人乳头瘤病毒(HPV)16晚期蛋白L1病毒样颗粒(VLPs)。方法构建HPV16L1基因序列优化前后的PET30aHPV16L1重组质粒,转化大肠杆菌Rosetta(DE3);用IPTG诱导目的基因表达,两步层析方法纯化HPV16L1蛋白;电镜下观察纯化产物形成VLPs的情况。结果成功构建大肠杆菌工程菌,高效可溶表达(目的蛋白约占总蛋白的38%)并纯化HPV16L1蛋白,纯度达95%以上,电镜下观察,发现纯化后的目的蛋白为直径50 nm左右,形态与天然病毒颗粒高度相似。结论在大肠杆菌原核系统中高效、简易地制备了HPV16L1VLPs,为诊断试剂和疫苗的研制奠定基础。     

Mapping of human serum-reactive epitopes in virus-like particles of human papillomavirus types 16 and 11

Most human antibodies against HPV16 can be blocked by the monoclonal antibody H16.V5. To investigate whether H16.V5 and human sera recognize similar epitopes, hybrid capsids containing different parts of HPV16 and HPV11 were evaluated for reactivity with human sera. The antibody responses among HPV 16-/HPV11+sera to HPV11 and to hybrid capsids containing the HPV11 C-terminus were strongly correlated. The antibody responses among HPV 16+/HPV11-sera to HPV16 and to a hybrid containing the HPV16 C-terminus were correlated and there was also reactivity with a hybrid containing the H16.V5 epitope in the HPV11 backbone. Several HPV16-/11- children's sera were reactive with hybrid capsids, implying that a native capsid structure is essential for serological specificity. For both HPV16 and HPV11, the major serologic reactivity was directed toward the C-terminal part of the protein and the H16.V5 binding site appeared to be a major serologically reactive epitope of HPV16.     

人乳头瘤病毒16型结构基因L1在昆虫细胞中的表达及其生物学活性

目的 研究与宫颈癌及人类其它多种组织癌症密切相关的人乳头瘤病毒16型（HPV16）的晚期基因L1的表达及其生物学活性。方法 采用PCR法从pBSSK-B/16L1扩增了来自中国妇女鲍温病组织标本的HPV16晚期基因L1,装入杆状病毒转移载体;在DH10Bac内通过转座子Tn7的介导,使携带有杆状病毒多角体蛋白基因启动子Ppolh的HPV16 L1基因整合入杆状病毒,形成重组杆状病毒;转染昆虫细胞Sf9进行表达;将感染72h的Sf9细胞包埋、切片、染色后,电镜观察。提取L1蛋白,免疫BALB／ c小鼠。结果 重组杆状病毒在Sf9细胞内高效表达出L1蛋白,经Western blot发现能与L1抗体特异地结合;薄层扫描显示所表达的L1蛋白占Sf9细胞总蛋白的比例高达31％。经透射电镜观察表明,在细胞核里有大量的重组杆状病毒形成;并且产生了大量的由HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明,所表达的HPV16 L1蛋白单体自组装的病毒样颗粒（virus-like particles,VLP)。小鼠免疫实验结果表明,所表达的HPV16 L1蛋白具有强免疫原性。结论 此研究为今后L1基因分子流行病学检查、L1蛋白的结构生物学研究、疫苗研制以及HPV相关基础性研究提供了有用的资料。     

Cell mediated immunity induced in mice by HPV 16 L1 virus-like particles

Recombinant human papillomavirus (HPV) type 16 L1 virus-like particles (VLPs) expressed in the baculovirus system were used to investigate the cellular immune response to human papillomavirus type 16. The cell-mediated immune response was evaluated through immunization of mice with HPV 16 L1 virus-like particles using a lymphoproliferation assay and cytokine production and cytometric analysis of lymphocyte subsets. A significant proliferative response was observed which was associated with secretion of both interferon-γ and interleukin-2. FACS analysis of splenic lymphocytes revealed that CD8⁺T-cells were increased in the immunized mice. These results demonstrate that HPV 16 L1 VLPs induce a T-cell response characterized by a Th1 profile and confirm that the HPV 16 VLP is a reasonable candidate for vaccine development.     

Self-assembly of in vitro-translated human papillomavirus type 16 L1 capsid protein into virus-like particles and antigenic reactivity of the protein.

The human papillomavirus type 16 (HPV-16) L1 capsid protein is the major component of the HPV virion. We prepared L1 protein of HPV-16 in a cell-free system. The L1 gene was cloned in an expression plasmid and transcribed and translated in vitro in a rabbit reticulocyte lysate. The expressed protein had the molecular mass (55 kDa) expected for the L1 protein, and it assembled into virus-like particles that closely resembled papillomavirus virions. The protein retained conformational epitopes, as evidenced by its reactivity with monoclonal antibodies which recognize only intact viral particles. In radioimmunoprecipitation assays with sera from college women grouped by their genital tract HPV DNA status, high reactivity was found in 68% of HPV-16 DNA-positive women, in 23% of women with other HPVs, and in 19% of HPV-negative women. In comparison, none of the sera of children were reactive. The results of the radioimmunoprecipitation assays showed a significant correlation with results obtained with the same sera in an enzyme-linked immunosorbent assay with virus-like particles produced in baculovirus (chi-square test for linear trend, P = 0.0023). Although the amounts of L1 protein obtained are small, the ability to produce virus-like particles by in vitro translation may be useful in the study of virus assembly, virus binding, and the immunological response to HPV infection.     

Physical interaction of human papillomavirus virus-like particles with immune cells

Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc gamma RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16(-) cell line with CD16 did not confer binding of VLP. Splenocytes from Fc gamma RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLP-receptor complex, possibly contributing to the immunogenicity of HPV VLP.     

Interaction of papillomavirus virus-like particles with human myeloid antigen-presenting cells

Papillomavirus-like particles (VLPs) are potent inducers of humoral and cellular immune responses, making them attractive candidates for noninfectious viral subunit vaccines. To further our understanding of how VLPs activate the immune system, we have investigated their interaction with human myeloid antigen-presenting cells. We found that VLPs bound, with increasing density, to the cell surface of human monocytes, macrophages, and monocyte-derived dendritic cells (DCs). Interestingly, there was a negative correlation between binding intensity and CD83 expression in DCs, suggesting that the main receptor for binding of VLPs may be downregulated during maturation. Exposure to VLPs resulted in acute phenotypic activation of monocytes and DCs. Furthermore, VLPs rapidly induced production of inflammatory cytokines in monocytes, macrophages, and DCs, as assessed by intracellular cytokine staining. For each cell type, the patterns of interleukin-1beta, interleukin-12, tumor necrosis factor-alpha, and interleukin-6 production were distinct from the pattern induced by lipopolysaccharide (LPS), a bacterial activator of myeloid antigen-presenting cells. Our results indicate that VLPs target multiple cells of the immune system, which helps to account for VLPs being so effective in priming humoral and cellular immune responses even in the absence of adjuvant.     

Cytokine and chemokine profiles following vaccination with human papillomavirus type 16 L1 Virus-like particles

To determine the systemic cytokine pattern induced by vaccination with human papillomavirus (HPV) L1 virus-like particles (VLP), we analyzed 22 different cytokines in culture supernatants of L1 VLP-stimulated peripheral blood mononuclear cells from vaccine (n = 19) and placebo (n = 7) recipients at months 0 and 2 after vaccination, using a multiplex cytokine bead array. In vaccine recipients, incubation with L1 VLP in vitro led to a statistically significant increase in production of Th1 (granulocyte-macrophage colony-stimulating factor, interleukin-2 [IL-2], gamma interferon; P < 0.0007) and Th2 (IL-4, IL-5, IL-10, IL-13; P < 0.0017) cytokines and the chemokine IP-10 (P = 0.0021) at month 2 after immunization, compared to levels seen prior to vaccination. These responses were not seen in placebo recipients. Cytokine and neutralizing antibody responses to vaccination followed the same pattern, with the highest antibody responses seen for subjects with higher cytokine responses. Cytokine profiling studies using samples from efficacy trials may provide important information about discriminators of long-term protection against HPV.     

Serological responses to human papillomavirus type 6 and 16 virus-like particles in patients with cervical neoplastic lesions.

Serum samples from 36 cervical carcinoma patients, 33 patients with high-grade squamous intraepithelial lesions, and 31 cytologically normal women were tested by enzyme-linked immunosorbent assay (ELISA) using human papilloma virus type 6 (HPV 6) and HPV 16 virus-like particles as antigens. Forty serum specimens from 1-year-old children were used to assign cutoff points. When serum samples from the subjects infected with HPV 16 were tested in an HPV 16 ELISA detecting immunoglobulin A (IgA), IgG, and IgM binding, 61% showed IgA, 44% showed IgG, and 39% showed IgM reactivity. Of HPV 6- or 11- or HPV 18-infected subjects. fewer than 17% showed IgA or IgG responses and 33% showed IgM reactivity. In contrast, 13% showed IgA, 10% showed IgG, and 16% showed IgM reactivity in the HPV DNA-negative controls. The results suggest that the IgA and IgG responses are HPV 16 specific and the IgM response is cross-reactive to different HPV types. On the other hand, the serological responses to HPV 6 did not differ in the patient and control groups. The percentages of patients positive for both IgA and IgG antibodies were significantly higher in the groups with high-grade squamous intraepithelial lesions (12% [4 of 33]; P = 0.04) and cancer (17% [6 of 36]; P = 0.02) than in the healty women (0% [0 of 31]), and the percentages for either IgA or IgG were higher for the cancer group (47% [17 of 36]; P = 0.01) than in the normal group (19% [6 of 31]). Most sera positive for IgA and IgG in the patient groups showed higher titers than those in the normal group. All these results suggest that high IgA and IgG responses are good indicators for estimating HPV 16 infection.     

Noninferiority of antibody response to human papillomavirus type 16 in subjects vaccinated with monovalent and quadrivalent L1 virus-like particle vaccines

The incorporation of multiple antigens into a single human papillomavirus (HPV) vaccine may induce immune interference. To evaluate whether interference occurs when HPV type 16 (HPV16) virus-like particles are combined in a multivalent vaccine, we conducted a study to evaluate anti-HPV16 responses among subjects receiving three-dose regimens of either a monovalent HPV16 vaccine or a quadrivalent HPV (types 6, 11, 16, and 18) vaccine.     

人乳头瘤病毒16型野毒株L1-E7融合基因重组腺病毒载体构建及在293细胞中表达嵌合病毒样颗粒的研究

目的制备人乳头瘤病毒HPV16L1-E7重组腺病毒,以期获得防治宫颈癌的重组腺病毒减毒活疫苗。方法以HPV16型的野毒株HPV16-114/K为模板,利用PCR克隆技术制备HPV16L1-E7融合基因pUC19L1-E7,含L1的1～301氨基酸（AA）和E7的1～60AA的编码DNA序列;并转入腺病毒穿梭质粒pCA14L1-E7,与腺病毒质粒pBHG10共转染293细胞,筛选重组腺病毒rAd5HPV16L1-E7。以PCR、ELISA和Westernblot方法鉴定重组病毒及其表达的L1-E7蛋白,密度梯度超速离心纯化HPV16嵌合L1-E7VLP,电镜观察VLP的形态结构。结果PCR-DNA序列分析表明成功构建了HPV16L1-E7重组质粒pUC19L1-E7;并获得HPV16重组腺病毒rAd5HPV16L1-E7,在293细胞中可高效表达L1-E7蛋白,并形成嵌合病毒样颗粒（cVLP）。结论构建的重组腺病毒rAd5HPV16L1-E7可有效表达HPV16L1-E7cVLP,可进一步用于动物实验,研究其在防治宫颈癌中的作用。     

Polymer-based enzyme-linked immunosorbent assay using human papillomavirus type 16 (HPV16) virus-like particles detects HPV16 clade-specific serologic responses

Human papillomavirus type 16 (HPV16) virus-like particles (VLP) were used as antigen in a polymer enzyme-linked immunosorbent assay (ELISA) to measure antibodies to HPV capsid proteins. Serum samples from 575 college women, previously tested for the presence of cervicovaginal HPV DNA, were analyzed. The prevalences of anti-HPV16 VLP antibodies at baseline were 14.1% for immunoglobulin G (IgG) and 6.4% for IgA. The seroprevalences of IgG in women with cervicovaginal HPV16, HPV16-related types, and other HPV types were 55, 33, and 19%, respectively (P < 0.001), compared to the prevalence in women without an HPV infection (10%). HPV VLP IgA seropositivity was associated with high HPV16 VLP IgG optical density values. The seropositivity of IgG antibodies was independently associated with infection with HPV16 or HPV16-related types, increased number of lifetime male partners for vaginal sex, having sex with men >/= 5 years older, history of abnormal PAP smear, older age, and living separately from parents. Use of HPV16 VLP polymer ELISA detects clade-specific responses and suggests an HPV16 VLP vaccine may have broader protection that initially anticipated.     

Immunogenicity against human papillomavirus type 16 virus-like particles is strongly enhanced by the PhoPc phenotype in Salmonella enterica serovar Typhimurium

Recombinant Salmonella strains have been widely used to deliver heterologous antigens and induce immune responses in vaccinated animals and humans. It remains to be established, however, how these bacteria mount an immune response; this has prevented the rational design of vaccines. Here we report for the first time that a particular genetic program, PhoPc, is necessary for recombinant Salmonella strains to induce an antibody response to a heterologous antigen, the human papillomaviruses type 16 (HPV16) virus-like particle (VLP). The PhoPc phenotype results from a point mutation in phoQ, the gene encoding the sensor component of a two-component regulatory system (PhoP-PhoQ) that controls the expression of a number of virulence factors in Salmonellae. To demonstrate that immunogenicity of the viral antigen expressed by the bacterial vector was dependent on the PhoPc phenotype, we have expressed the phoQ mutant gene (phoQ24) in two differently attenuated Salmonella enterica serovar Typhimurium strains. Our data show extrachromosomal phoQ24 to be dominant over the chromosomal copy of the phoQ gene, conferring the PhoPc phenotype on the recipient strains. In addition, activation of PhoPQ-regulated genes by the plasmid-encoded PhoQ24 did not alter bacterial survival and conferred immunogenicity to the HPV16 VLP expressed in the two S. enterica serovar Typhimurium backgrounds, inducing the production of HPV-specific antibodies in mice. This strongly suggests that at least one of the PhoP-regulated genes is necessary for mounting an efficient antibody response to HPV16 VLP. This finding sets the stage for further development of a Salmonella-based vaccine against HPV infection and cervical cancer.     

重组人乳头瘤病毒6型病毒样颗粒的免疫原性分析

目的　研究重组人乳头瘤病毒 6型 (humanpapillomavirustype 6 ,HPV 6 )病毒样颗粒(virus likeparticle ,VLP)的免疫原性。方法　重组杆状病毒在昆虫细胞中表达制备的HPV 6L1VLP(L1 VLP)和HPV 6L1+L2VLP(L1+2 VLP)经鉴定后 ,用于免疫BALB/c小鼠 ,对诱导的体液免疫和细胞免疫反应进行了检测。结果　电镜观察显示L1 VLP和L1+2 VLP二者形态上无明显差异 ,为圆形颗粒 ,直径约 5 0nm ,SDS PAGE和Westernblot分析表明 ,L1+2 VLP中L1和L2蛋白摩尔比例为 4∶1。用ELISA法测定免疫小鼠血清抗体滴度 ,加佐剂L1 VLP免疫组和加佐剂L1+2 VLP免疫组血清针对HPV 6L1VLP的滴度在 1∶10 0 0 0以上 ,高于未加佐剂组免疫血清滴度 (1∶2 0 0 0 ) ,L1+2 VLP免疫诱导出了特异于L2抗原的抗体。血清抗体主要识别HPV 6构象依赖性抗原表位 ,与HPV 11抗原显示出一定的交叉反应 ,而与HPV 16无明显交叉反应。免疫小鼠脾淋巴细胞体外经HPV 6L1VLP再激活后出现了特异性增殖反应 ,3H TdR掺入值与未免疫组之间差异有显著性 (P <0 .0 1) ,L1 VLP和L1+2 VLP两组间差异无显著性 (P >0 .0 5 ) ,L1 VLP和L1+2 VLP免疫组刺激指数 (SI)分别为 6 .4和 6 .2 ,阴性对照组SI为 1.1。HPV 6L1VLP再刺激特异地诱导免疫组脾淋巴细胞IL 2和IL 10分     

Enhanced mucosal and systemic immune responses following intravaginal immunization with human papillomavirus 16 L1 virus-like particle vaccine in thermosensitive mucoadhesive delivery systems

To develop more potent and convenient mucosal human papillomavirus (HPV) vaccines, we tested the effect of thermosensitive mucoadhesive vaginal vaccine delivery systems on the local and systemic antibody responses to HPV 16 L1 virus-like particles (VLP). HPV 16 L1 VLP expressed from recombinant baculovirus-infected Sf21 insect cells were delivered in phosphate-buffered saline (PBS) or thermosensitive mucoadhesive delivery systems, composed of poloxamers (Pol) and varying amounts of polyethylene oxide (PEO). Pol/PEO-based vaginal vaccine delivery systems existed in liquid form at room temperature, but gelled at 37 degrees C. The mucoadhesiveness of Pol/PEO-based delivery systems increased with PEO, but the formulations with PEO higher than 1.0% were too viscous to be administered into the vagina. Vaccine vehicles affected the vaginal and salivary immune responses to HPV 16 L1 VLP intravaginally administered into mice. At 42 days after the first intravaginal immunization of HPV 16 L1 VLP with cholera toxin, vaginal and salivary IgA titers were the highest in the group given in Pol/PEO 1.0% vehicle followed by Pol/PEO 0.4% and PBS vehicles. Intravaginal coadministration of HPV 16 L1 VLP and cholera toxin in Pol/PEO 1.0% showed 31- and 39-fold higher titers compared to the PBS-based HPV 16 L1 VLP groups administered by intravaginal and intramuscular routes, respectively. Following intravaginal administration, Pol/PEO 1.0%, but not Pol/PEO 0.4%, showed significantly higher HPV 16 L1 VLP-specific serum IgG titers as compared to the PBS vehicle. Our results indicate that the use of in situ-gelling vaginal vaccine delivery systems with increased mucoadhesiveness would be beneficial for more effective induction of mucosal and systemic immune responses to intravaginally administered HPV 16 L1 VLP vaccines.     

Human papillomavirus type 58 L1 virus-like particles purified by two-step chromatography elicit high levels of long-lasting neutralizing antibodies

Human papillomavirus (HPV) type 58 is a high-risk type of HPV frequently detected in cervical cancers, especially in Eastern Asia. There are still no commercially available vaccines against HPV 58 infection. High levels of long-lasting neutralizing antibodies are crucial for long-term protection against HPV infection. Here, we have developed a two-step chromatography strategy and have purified highly pure HPV L1 proteins, which form more homogenous and uniform VLPs than those purified by CsCl ultracentrifugation. Low-dosage immunization with HPV 58 L1 VLPs alone or co-administrated with HPV 16 and HPV 18 L1 VLPs is sufficient to induce high levels of long-lasting neutralizing antibodies in mice. Our results suggest that the highly immunogenic HPV 58 L1 VLPs are a good candidate for use in developing effective vaccines against HPV 58 infection.     

A human papillomavirus type 16 vaccine by oral delivery of L1 protein

To establish an edible HPV16 vaccine, we constructed a recombinant HPV16 L1-expressing Schizosaccharomyces pombe yeast strain (HPV16L1 yeast). A preliminary study revealed that freeze-dried yeast cells could be delivered safely, and were digested in the mouse intestine. The freeze-dried HPV16 L1 yeast was administered orally as an edible vaccine, with or without the mucosal adjuvant heat-labile toxin LT (R192G), to 18 female BALB/c mice. After the third immunization, none of the mice that received the edible HPV16 vaccine showed specific antibody responses, whereas all of the positive controls that were administered intranasally with 5 μg of HPV16-virus-like particles (VLP) had serum IgG, and genital IgA and IgG that reacted with HPV16-VLP in enzyme-linked immunosorbent assays (ELISAs). When a suboptimal dose (1 μg) of HPV16-VLP was administered to all the mice, including the negative control mice, 50% of the mice that were pre-immunized with the edible HPV16 vaccine showed positive serum IgG responses, while none of the negative controls showed any response. Vaginal IgG and IgA antibodies were also elicited in 33 and 39%, respectively, of the mice that were given with the edible HPV16 vaccine and the intranasal boost. All of the antibodies reacted more strongly to intact HPV16-VLP than to denatured HPV16-L1 protein suggesting that the edible vaccine primes for antibody responses against conformation-dependent epitopes. The inclusion of adjuvant in the vaccine formulation marginally increased the genital IgA response (P = 0.06). HPV16-L1 protein in the yeast might induce tolerance in the vaccinated animals that could be recovered by intranasal boosting with a suboptimal dose of HPV-VLP. This freeze-dried yeast system may be useful as an oral delivery of HPV 16 L1 protein.     

Tetramethylbenzidine staining procedure after starch gel electrophoresis of human haemoglobin

The purpose of the present work was to increase the stability of tetramethylbenzidine-stained electrophoretic patterns of human haemoglobin through use of a suitable fixing agent. Since haemoglobin examination is quite important in the study of numerous pathologies, in prenatal analyses and in forensic medicine, it was considered worthwhile to attempt to stabilise the specific tetramethylbenzidine-based stain, which is notoriously unstable. The other stain frequently used is amido black 10 B, a generic protein stain, which reveals both haemoglobin and non-haemoglobin bands, hence interpretation of the results can lead to errors and the identification of false pattern heterogeneity. Following several experimental tests we have observed that it is possible to make tetramethylbenzidine-based stains stable by using a suitable fixative. Treatment with the fixing solution prevents the colour from fading, and can keep it intact for over a year. The method also appears promising for studying haemoglobin in a variety of other species of vertebrates.     

Tetramethylbenzidine staining procedure after starch gel electrophoresis of human haemoglobin

The purpose of the present work was to increase the stability of tetramethylbenzidine-stained electrophoretic patterns of human haemoglobin through use of a suitable fixing agent. Since haemoglobin examination is quite important in the study of numerous pathologies, in prenatal analyses and in forensic medicine, it was considered worthwhile to attempt to stabilise the specific tetramethylbenzidine-based stain, which is notoriously unstable. The other stain frequently used is amido black 10 B, a generic protein stain, which reveals both haemoglobin and non-haemoglobin bands, hence interpretation of the results can lead to errors and the identification of false pattern heterogeneity. Following several experimental tests we have observed that it is possible to make tetramethylbenzidine-based stains stable by using a suitable fixative. Treatment with the fixing solution prevents the colour from fading, and can keep it intact for over a year. The method also appears promising for studying haemoglobin in a variety of other species of vertebrates.