| Pine pollen polysaccharide has the very high nutritional value, and our group has beenpreliminarily proved that the Taishan pine pollen polysaccharide has the function ofenhancing immunity. Compared with some chemical synthesis immunopotentiator, Pinepollen polysaccharide has many virtues, such as a variety of sources, low toxic and side effect,no carcinogenic effect, high safety and so on. Lactobacillus is recognized as the microbialadditives with the world’s longest history and found earliest in application. Lactobacillus isone of the animal intestinal advantage bacterium groups, numerous studies have shown that itcan improve the body metabolism, promote intestinal bacteria proliferation, inhibit the growthof harmful bacteria and increase the effect of immune. A series of problems produced byantibiotic gradually draw people’s attention, and researching new safe and effective immuneenhancers become the need of the development of animal husbandry. Pine pollenpolysaccharide and Lactobacillus both have a certain immune enhancement effect, but thecommon study of both at home and abroad had not be reported. The B.avium outer membraneprotein (OMP) was extracted by using ultrasonic fragmentation, TritonX-100processingtechnology, etc. Its content was determined by the Bradford method and it was detected bySDS-PAGE. The polysaccharides of fresh pine pollen were obtained through water extractionand ethanol precipitation. Last, the subunit vaccine with the polysaccharide level of100mg/mland the B.avium OMP level of750μg/ml and the B.avium OMP subunit vaccine with nopolysaccharide are prepared for sterility as well as safety testing. This research consists ofthree parts as follows:1The synergistic effect of Taishan pine pollen polysaccharide andLactobacillus in enhancing the immunity of subunit vaccine of B.aviumOMPIn order to research the effect of immune enhancement caused by Taishan pine pollenpolysaccharide (TPPPS) and Lactobacillus. Three hundred one-day-chicks were randomlydivided into six groups, group I, II, III, and IV were immuned by subunit vaccine of B.aviumOMP contains TPPPS at100mg/ml, group VI were immuned by subunit vaccine of B.aviumOMP without TPPPS. Group I and II were respectively orally given one milliliter of high andlow dose of live Lactobacillus. In the first two weeks, group III and IV were respectivelyorally given one milliliter high and low doses of heat-killed Lactobacillus in the first two weeks. Samples of blood and small intestine were taken every week after the firstimmunization. The level of systemic antibody was detected by indirect ELISA method,peripheral blood lymphocyte ratio was detected by automated hematology analyzer,peripheral blood lymphocyte transformation rate was detected by MTT method, intestinalsIgA levels and serum IL-2levels were detected by ELISA method. The results showed thateach index in group I, II, III and IV were significantly higher than that in group V and VI（P<0.05）; each test index in group V were significantly higher than that in group VI（P<0.05）; each index in the groups of live Lactobacillus were slightly higher than that in thegroups of heat-killed Lactobacillus, each test index in high dose groups were slightly higherthan that in low dose groups. Therefore, pine pollen polysaccharide can significantly improvethe immune effect of vaccine as the immune adjuvant, and oral administration ofLactobacillus can further enhance the animal immune response ability to the vaccine, thedifferences of each test index between oral administration of live Lactobacillus and oraladministration of heat-killed Lactobacillus were not significant. Pine pollen polysaccharideand Lactobacillus have the synergistic effect in enhance the immunity of subunit vaccine ofB.avium OMP.2The influence of Pine pollen polysaccharide on the vitro growth ofLactobacillus and study the antiviral effectThe modified method of water extraction and alcohol precipitation of Ma Rongchi Etc.was used to extract high-dose TPPPS, and TPPPS of different concentrations were separatelyprepared. The same dose of Lactobacillus were put in the TPPPS culture medium at differentconcentration and test the absorbance (A) values (360nm) of the bacterial culture mediumcontaining different concentration of TPPPS which has been diluted10times and that ofcontrol group at0,18and24h. Results showed that TPPPS could promote the growth ofLactobacillus, and the best work concentration was10mg/ml.TPPPS, two co-culture were filtered bacteria. Then detection the antivirus effect of thethree group using SPF chicken embryo inoculation method, results showed that The HA resultof two co-culture groups was lower than TPPPS stimulus alone group, and experimentalgroups were lower than virus control group. With the increase of action time, HA resultsbecame lower.3Synergy of Lactobacillus and Taishan pine pollen polysaccharide onenhancing immunity in Mice This study was to examine the synergy of immunological enhancement of Lactobacillusand TPPPS on Mouse immunized with B.avium OMP subunit vaccine. In vitro, the effect ofTPPPS at different dosages on the growth of Lactobacillus was determined, then lymphocyteproliferation as well as IL-2secretion was observed. In vivo experiment,240clean-levelKunming mice (female) were randomly divided into four groups (I~IV). In the first twoweeks, group I were fed with Lactobacilli, group II were fed with Lactobacilli and TPPPS,group III were fed with TPPPS, group IV were the control group. On the third day, all groupswere injected B.avium OMP subunit vaccine. Each week after immunization, serumantibodies, lymphocyte ratio, intestinal sIgA and serum IL-2levels was detected. The resultsshowed that, in vitro, The TPPPS and Lactobacillus culture medium can both promote thelymphocyte proliferation and IL-2secretion, but the effect of their co-culture medium was themost significant (P<0.05). The vivo experiment proved that in most of the detection timepoint, each indicator of group I, II and III was significantly higher than that of group IV(control group)(P<0.05), and each indicator of group II was significantly higher than that ofgroup I and III(P<0.05). Results suggested that Lactobacillus and TPPPS have a synergisticeffect on improving the immunity of mouse. |
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