| ObjectiveTo understand the trends of China’s AIDS and its three distribution characteristicsover the past ten years, and provide clues for HIV/AIDS prevention. To explore therole of complement in the pathogenesis of AIDS and its mechanism, and perform theexperiment of the activation of protein CR2-IgG1Fc on local complement (laterabbreviated CR2-Fc) to inhibit HIV infection, providing a new idea for the preventionand control ofAIDS.Methods1. Descriptive analysis(1) Database Construction2004-2013China’s31provinces (municipalities, autonomous regions) of AIDSmorbidity and mortality data of epidemic situation (excluding Hong Kong, Macao, andTaiwan) from Chinese diseases reporting information system; the provinces populationdata comes from the national population and Health Sciences sharing platform;1:4,000,000across the province (municipalities, autonomous regions) level electronicmap comes from National Geographic Information Research institute. Then ttheepidemic situation report data and population data were classified and screened intoExcel2010.(2) MethodsWe adopt descriptive epidemiological analysis method to analyze the AIDSepidemic trend of2004-2013in China and ArcGIS software to draw the exhibitionspace dynamic change of China’s AIDS incidence. We mainly described the threedistribution characteristics and trend of the main description ofAIDS.(3) Statistical analysisData from Excel2010formed a database. SAS9.2was used as to study the incidence, mortality and index constituent ratio. χ2test was used to compare the rate andconstituent ratio. Charts were drawn with Excel2010and Prism5.0software.2. Expression, activity detection and inhibition of HIV infection of local complementactivation protein CR2-Fc(1) Construction and expression of CR2-FcThe PCR amplified fragments of SCR1-4from CR2gene and IgG1Fc fragmentswhich were connected and constructed into T vector. XbaⅠand NotⅠwere used todigest the recombinant T vector and pCI-neo eukaryotic expression vector, T4ligasewasused to connect CR2-Fc gene into the eukaryotic expression vector, to obtainrecombinant eukaryotic expression vector pCI-neo/CR2-Fc. Recombinant eukaryoticexpression vector was transfected into CHO K1cells, G418screening to obtain stableexpression cell lines. Expression of CR2-Fc protein was detected by RT-PCR andWestern blot. Stably expressing monoclonal cell lines were cultured in serum freemedium, application of proteinG agarose affinity column to purify target protein in thesupernant. Detection of purified CR2-Fc by SDS-PAGE and BCA protein quantitativemethod.(2) Protein activity detection and the inhibition of HIV-1infectionSheep red blood cell lysis experiments were used to determine the ability ofCR2-Fc to activate the complement. CR2-Fc in gradient dilution was added intoreaction system where complement is easily activated. Detect the dose effectrelationship between CR2-Fc and complement activation ability. Application ofanti-CR2mAb (clone1048) to block C3d binding sites, observe the hemolytic changes.Flow cytometry was used to detect the target of CR2-Fc binding, namelyCR2-Fcbinding to cell surface C3d, APC labled anti-C3d mAb markers and FITC labledanti-CR2mAb were added to detect double fluorescence signal in the cell surface. UsePBS instead of CR2-Fc to block C3d deposition, detect cell surfacefluorescencechanges respectively.Tzm-BL cell fluorescence method to indirect assessment of killing capacity of freeHIV. Infection HIV-1of Tzm-BL cells containing fluorescent marker gene, thefluorescence intensity of Tzm-BL cells infected with HIV was proportional to theamount of HIV, and the inhibition ability was inversely proportional. By adding different concentrations of CR2-Fc into HIV-1with human serum, determine therelationship between CR2-Fc and fluorescence intensity, and estimation indirectly theability of recombinant protein to inhibite HIV.ATP fluorescence method by estimatingthe number of live cells to judgethe ability of CR2-Fc to lysis cells. Differentconcentration of CR2-Fc and the amount of serum were added into HIV-1infected H9cells, ATP fluorescence were examined after incubating for4h. Fluorescence values canbe used to estimate the ability of CR2-Fc adsorption and killing of HIV cells.Results1. Analysis descriptive epidemiological status of the AIDS epidemic in Chinain2004-2013.(1) In the past decade, China’s AIDS epidemic is increasing year by year, both theincidence and mortality increased(Cochran-Armitage trend testfor incidence, Z=295.27,p<0.05;Cochran-Armitage trend testfor mortality, Z=154.06，p<0.05). The morbidityincreased from0.28/100,000in2004to3.06/100,000in2013, with an increase by992.86%; the death rate increased from0.06/100,000in2004to0.83/100,000in2013,with an increase by1283.33%.(2) The distribution of AIDS was wide and had large regional differences. All31provinces (municipalities, autonomous regions) had AIDS case report. Provinces withdisease rate higher than1/100,000from1in2004rose to21in2013. The highincidence of AIDS mainly concentrated in the southwest of China. The average AIDSannual incidence of Guangxi province with rate of6.51/100,000ranks first in thecountry, the other provinces with high incidence are Yunnan, Xinjiang, Henan, with theannual incidence rate of5.29/10million,3.89/10million,2.10/10million respectively.The3provinces with lower average annual incidence rate are Shandong province0.12/100,000, Inner Mongolia0.13/100,000and Tibet0.16/100,000, respectively.(3) Male patients of AIDS were more than female in the year2004-2013, male patientsaccounted for70.48%of the total number of cases, the male to female ratio was3.88:1.In last decade, male and female patients ratio increased year by year (Cochran-Armitagetrend test, Z=32.17, p<0.05), the ratio increased from1.92:1in2004to2.97:1in2013.The incidences of AIDS mainly focus in young population, cases aged20-59accountedfor86.35%of the total cases. The30-39age groupwas the high incidence of AIDSconsidering age,32.38%of total reported cases, followed by40-49age group of21.67% and20-29age group of20.57%.0-9years cases accounts for least, accounted for only1.12%.2. Local complement activation protein CR2-Fc’s inhibition of HIV infection.(1) The recombinant eukaryotic expression vector construction, expression andpurification ofCR2-Fc protein.Synthesis of CR2(SCR1-4) gene and human IgG1Fc gene were conducted by PCR,and1%agarose gel electrophoresis showed that CR2-Fc gene band at1800bp; Afterdouble enzyme digestion of the recombinant eukaryotic expression vectors,electrophoresis showed two bands in5800bp and1800bp, consistent with the expectedresults; recombinant eukaryotic expression vector was sended to Shanghai SANGONbiological engineering Limited by Share Ltd to sequence. The results of sequencingwere compared with Genbank, showing that the target gene is accurate. Extraction oftotal mRNA in the transfected cells with Trizol, RT-PCR showed:1%agarose gelelectrophoresis showed a band of about1800bp, consistent with expectations, andproved the existence of intracellular CR2-Fc transcription. The anti-CR2mAb(clone1048) and anti-IgG1Fc mAb were used as the detection antibody in Western blot,results showed: reduced sample is located at60kDa, consistent with the expected results;non-reducing condition sample is located at130kDa, indicating the existence of CR2-Fctetramers under the natural conditions. Empty vector transfected supernatant was usedas control and showed no band; after purification of samples for SDS-PAGE testing,results showed: the reduced sample is located at60kDa; non-reducing sample is about130kDa, consistent with the Western blot.results. Quantity One software was used toanalysis the result of SDS-PAGE, indicated that the sample purity was greater than90%.BCA method was used for the quantitative detection of the purified protein, thecalculated CR2-Fc protein concentration was about900μg/ml.(2) CR2-Fc activity determination and the inhibitionof HIV infectionThe hemolysis test: complement activation to form MAC could induce erythrocytelysis. CR2-Fc in gradient dilution was added in the reaction system and we found that:with the increasing of CR2-Fc concentration, red blood cell lysis rate increasedsignificantly (Cochran-Armitage trend test, Z=2.12, p<0.05). An overexpression ofanti-CR2mAb (clone1048,250μg/ml) could competitively inhibite CR2-Fc and C3dcombination, the complement activating ability CR2-Fc was significantly inhibited, the hemolysis rate was not significantly changed. The flow cytometry showed that normalactivation of complement and the addition of CR2-Fc molecules, we could detect theAPC and FITC double fluorescence signal in the cell surface; in the case of normalactivation of the complement, replace the CR2-Fc molecules with PBS, we could onlydetect APC signalon the cell surface;42mM EDTA could block complement activation,noAPC and FITC signal could be detected.Detection of CR2-Fc by Tzm-BL cell fluorescence method on free HIV killingability: In the presence of low concentrations of neutralizing antibodies, recombinantprotein CR2-Fc can target C3d molecules on the surface of HIV-1amplification theactivation of the complement, and speed the virus dissolution. With the increaseconcentration of CR2-Fc molecules, the HIV-1killingrate was increased. When theconcentrationof CR2-Fc reached to10.31μM,virus lysis rate reached the peak of34.26±6.43%. To replace NHS with C6deficient serum, there isno obvious change of thefluorescence signal.The ATP fluorescence method showed thatwith the increase in theconcentration of dissolved molecules, the ability to lysis infected cells was enhanced,with cell lysis rate increased from-2.10±3.52%(CR2-Fc concentration of0.01μM) to18.49±4.28%(CR2-Fc concentration of10.31μM). With C6deficient serum ratherthan normal human serum, fluorescence detection results showed no obvious change.ConclusionsIn the past decade, the morbidity and mortality of China’s AIDS were stillincreased and the distribution of AIDS is wide and has large regional differences, theprevention and control of the situation is not optimistic. Considering the role andcharacteristics of complement in HIV infection, we designed and expressed thelocalcomplement activation protein CR2-Fc. Experiments confirmed that CR2-Fc had goodbiological activity and the capability to inhibite HIV-1infection. |
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